"We will seek an explanation from Russia on why it took this action and assurances this will not happen again," says a US defence offcial.
Russian warplanes struck at US-backed forces battling DAESH terrorists in eastern Syria on Thursday, a senior US defence official said.
The official, who spoke on condition of anonymity, criticized the Russian air strikes near Al Tanf border crossing with Iraq.
He said no Russian or Syrian ground forces were in the area at the time.
"Russia's latest actions raise serious concern about Russian intentions," the official said.
"We will seek an explanation from Russia on why it took this action and assurances this will not happen again."
British-based monitoring group the Syrian Observatory for Human Rights (SOHR) said warplanes had struck a meeting of New Syria Army (NSA) near the Al Tanf killing two fighters and wounding four others. The NSA is US-backed opposition forces consists of fighters mostly from the provinces of Homs and Deir Ezzor, who were trained by the forces of the international coalition in Jordan camps and they were provided with advanced military equipment.
However, the SOHR said that "the warplanes that targeted the meeting were not identified yet."
Washington has consistently refused to join forces with Russia in Syria against DAESH ever since Moscow launched its campaign of air strikes in September last year, accusing it of acting solely to prop up leader of Syrian regime Bashar al Assad.
Dozens of State Department employees have endorsed an internal document that advocates US military action to pressure Syria's government into accepting a cease-fire and engaging in peace talks, officials said Thursday.
Obama called for regime change early on in the conflict and threatened military strikes against Syrian regime forces after using chemical weapons in 2013. But Obama only has authorized strikes against DAESH and other US-designated terror groups in Syria.
The UN estimates that the death toll in Syria during the five-year-long war is at least 250,000. But the Syrian Centre for Policy Research released a report on February this year, saying that the death toll has now exceeded 470,000.
Around 5 million others took refuge in the neighbouring countries of Turkey, Lebanon, Iraq and Egypt, with Turkey hosting the largest number at almost 3 million.