"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.