According to the Russian Defense Ministry, warplanes targeted eight ISIS positions, including arms, transportation, communications and control positions.
But U.S. Defense Secretary Ash Carter countered that claim.
"I want to be careful about confirming information, but it does appear that they (Russian airstrikes) were in areas where there probably were not ISIL forces," he told reporters. ISIL is an acronym for ISIS.
"The result of this kind of action will inevitably, simply be to inflame the civil war in Syria," Carter said.
A senior U.S. administration official told CNN's Elise Labott that a Russian airstrike near the Syrian city of Homs "has no strategic purpose" in terms of combating ISIS, which "shows they are not there to go after ISIL."
Syrian state-run news agency SANA
reported that Russian warplanes had targeted "ISIS dens" in al-Rastan, Talbiseh and Zafaraniya in Homs province; Al-Tilol al-Hmer, in Qunaitra province; Aydoun, a village on the outskirts of the town of Salamiya; Deer Foul, between Hama and Homs; and the outskirts of Salmiya.
According to the London-based Syrian Observatory for Human Rights, 28 people were killed in the strikes, including women and children. The Syrian National Coalition reported that 36 people were killed, all civilians.
The U.S. official said the United States had no intention of preventing the strikes, but that Russian planes didn't seem to be flying in areas where the United States is operating.
"They are not stupid," the official said.
Kerry: 'Grave concerns'
U.S.-led coalition missions were continuing as normal despite an advance warning and request from Russia to stay out of Syrian airspace.
During a statement at the U.N. Security Council meeting on fighting terrorism, U.S. Secretary of State John Kerry said: "We have conducted a number of strikes against ISIL targets in Syria over the past 24 hours including just an hour ago. These strikes will continue."
Kerry said the U.S.-led coalition had conducted 3,000 airstrikes against ISIS and that efforts would dramatically increase.
He warned that the fight against ISIS should not be confused with support for Syrian President Bashar al-Assad.
"Moreover, we have also made clear that we would have grave concerns should Russia strike areas where ISIL and al Qaeda affiliated targets are not operating. Strikes of that kind would question Russia's real intentions -- fighting ISIL or protecting the Assad regime," he said.
Later, Kerry and his Russian counterpart, Sergey Lavrov, said that military talks between their two countries would happen soon.
"The first instruction to us was to make sure that the military of the United States, the coalition led by the United States on one hand, and the military of the Russian Federation who now engage in some operations in Syria at the request of the Syrian government -- get in touch and establish channels of communications to avoid any unintended incidents," said Lavrov.
A senior U.S. defense official told CNN the Pentagon was "taken aback" by Russia's actions. "Our Presidents just talked about setting up de-confliction talks and now they just go ahead and do this? They cannot be trusted."
Another U.S. official said: "This is not how military relations are conducted."
Pentagon spokesman Peter Cook had told reporters that Secretary Carter had directed his staff to "open lines of communication with Russia on de-confliction." The purpose of discussions would be "to ensure the safety of coalition air crews," he said.
Russia: Coalition strikes on ISIS illegal
Earlier Wednesday, the upper house of the Russian Parliament gave President Vladimir Putin approval to use the air force in Syria, state media reported.
"The Federation Council unanimously supported the President's request -- 162 votes in favor of granting permission," Kremlin Chief of Staff Sergey Ivanov said, according to state-run ITAR-Tass news agency.
Federation Council Speaker Valentina Matviyenko said that the Assad regime was the only legitimate force fighting ISIS, ITAR-Tass reported
. It quoted her as saying that strikes by the U.S-led coalition violated international law as "interference into the territory of a sovereign state can only be carried out on authorization of U.N. Security Council or on request of official legitimate authorities."
Matviyenko's comments were echoed by Kremlin spokesman Dmitry Peskov, ITAR-Tass reported
. "As a matter of fact Russia will be the sole country that will be carrying out that operation on the legitimate basis at the request of Syria's legitimate authorities," Peskov said.
Lavrov said Russia conducted airstrikes after a request from al-Assad.
Speaking at the start of the U.N. Security Council meeting to combat terrorism, Lavrov said: "On the 30th of September in response to a letter by the President of Syria, the President of Russia asked and received the consent of the Council of Federation for the use of the armed forces of the Russian Federation in the Syrian Arab Republic."
He continued: "We're referring here exclusively to the operation of the Russian air force to carry out strikes against ISIL positions in Syria. We have informed the authorities in the United States and other members of the coalition created by the Americans of this and are ready to forge standing channels of communication to ensure maximally effective fight against the terrorist groups."
Israeli officials said Russia had contacted Israeli defense officials prior to conducting its airstrike operation in Syria.
French Foreign Minister Laurent Fabius said France had not received advance warning and he wanted to be sure Russia did not target opponents of the Assad regime or civilians.
"As far as the strikes themselves are concerned, we have to check that it really was Daesh and terrorist groups that really have been targeted and not opponents to the Syrian regime or the civilian population," Fabius told reporters, after giving a statement to the Security Council. Daesh is the Arabic acronym for ISIS. "I'm not accusing anybody of anything but we have to check the facts," he said.
'No Russian boots on the ground'
Putin, speaking at a government meeting, said his country would not become mired in the Syrian conflict.
"This military operation is limited in time. Russian air forces will help Assad's army while it's on the offensive mode," Putin said. "There will be no Russian boots on the ground."
Ahead of Wednesday's strikes, Russia's air force carried out several days of familiarization flights, and the collection of potential targeting information by drones.
Four Russian Su-34 Fullback fighter jets are now at the Latakia air base in Syria, and more than 600 Russian troops are in place, a U.S. official with knowledge of the latest intelligence told CNN this week.
U.S. officials previously have said Russia's movements suggest that its targets might be something other than ISIS.
"We see some very sophisticated air defenses going into those airfields. We see some very sophisticated air-to-air aircraft going into these airfields. I have not seen ISIL flying any airplanes that require SA-15s or SA-22s (Russian missiles). I have not seen ISIL flying any airplanes that require sophisticated air-to-air capabilities," Gen. Philip Breedlove, NATO's supreme allied commander, said on Monday.
"I'm looking at the capabilities and the capacities that are being created and I determine from that what might be their intent. These very sophisticated air defense capabilities are not about ISIL. They're about something else," he concluded.
Defense officials have previously told CNN that the United States believes Moscow may fear that al-Assad may not be able to retain power in the war-torn country and wants to be in position to be able to support a proxy should the situation collapse.
Russia is a close ally of al-Assad and may want to bolster him, while the United States has repeatedly called for him to go in order to resolve the five-year civil war.