"Today, Russian aircraft conducted a series of air strikes near al-Tanf against Syrian Counter-ISIL forces that included individuals who have received US support," said the senior defense official, who requested anonymity.
"Russian aircraft have not been active in this area of southern Syria for some time, and there were no Syrian regime or Russian ground forces in the vicinity," the official said.
It was not known how many fighters were struck and the extent of casualties or which group they belonged to.
The US military launched a $500 million program in early 2015 to train entire units of "moderate" Syrians to fight Islamic State jihadists.
But the program drew heavy fire last fall after admitting the efforts had floundered, with numbers of trainees falling massively short of the planned 5,000.
One group even handed over ammunition and other gear to a local Al-Qaeda affiliate, known as the Al-Nusra Front.
Since then, the Pentagon's new strategy is to work with just a handful of members from each fighting group, instead of an entire unit.
Signup to our newsletter and follow us on Facebook and Twitter!
Much of the attention is being focused on the Syrian Democratic Forces, a largely Kurdish coalition that has scored some significant gains against IS jihadists.
The CIA has also been involved in training Syrian rebels, though the secretive agency has not officially provided any details of its efforts.
The bombing would likely further strain already testy ties between Moscow and Washington on the Syrian issue.
"Russia's latest actions raise serious concern about Russian intentions. We will seek an explanation from Russia on why it took this action and assurances this will not happen again," the defense official said.
Russia and the United States co-chair a 22-nation group that supports a UN-led process to end Syria's five-year civil war through a negotiated deal.
On Wednesday, US Secretary of State John Kerry told Russia and Syrian President Bashar al-Assad to respect a fragile ceasefire, warning that Washington's patience was running out.
World powers have failed to turn the cessation of hostilities, in effect since February 27, into a durable truce and Damascus has stepped up its military campaign against the Islamic State group and rebels, especially in the city of Aleppo.
The United States has accused Russia of working to consolidate the regime of Assad, its ally, and continuing to attack the opposition.
The five-year war has killed more than 270,000 people and displaced millions.