The official, speaking on condition of anonymity, told reporters on Thursday that Russia bombed US-backed militants in southern Syria earlier in the day, raising "serious concern" over the incidence.
"Today, Russian aircraft conducted a series of airstrikes near al-Tanf against Syrian counter-ISIS forces that included individuals who have received US support," said the senior defense official.
"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."
"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.
The US military launched a $500 million program in early 2015 to train entire units of what it described as "moderate" militants in the Arab country. 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 has worked with militants from different groups, instead of an entire unit.
This is while the CIA has also been involved in training militants in Syria.
Since September 2014, the US and some of its allies have been carrying out airstrikes against purported positions of Daesh (ISIL) militants inside Syria.
The Takfiri militants, who were initially trained by the CIA in Jordan in 2012 to destabilize the Syrian government, control large areas of Iraq and Syria.
The Syrian government has charged that the airstrikes had targeted the country's infrastructure in many instances and done little to stop the advances of terrorists.
Syria’s years-long foreign-sponsored militancy has claimed hundreds of thousands of lives since early 2011 and left behind vast trails of destruction in a country where millions of people have been displaced, Press TV reported.
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