The United States is following "extremely disturbing accounts" of large numbers of people killed in a village in central Mali, State Department spokesperson Ned Price said on Sunday.
Mali's military said on Saturday that it had killed more than 200 Islamist militants in the latest clash in a month of escalating violence.
Tensions with the West have increased since a move by Mali's ruling military junta to delay democratic elections in February, as well as over its collaboration with private military contractors belonging to Russia's Wagner Group.
In a statement, Price said there were conflicting reports about who was responsible for the killings that took place in late March in the village of Moura, about 250 miles (400 km) northeast of the capital, Bamako.
"We are concerned that many reports suggest that the perpetrators were unaccountable forces from the Kremlin-backed Wagner Group. Other reports claim the Malian Armed Forces (FAMa) had targeted elements of known violent extremist groups," he said.
"These conflicting reports illustrate the urgent need for the Malian transition authorities to give impartial investigators free, unfettered, and safe access to the area where these tragic events unfolded."
Officials at Russia's embassy in Washington declined immediate comment on reports of the Wagner Group's involvement.
The European Union has imposed sanctions on the Wagner Group, accusing it of clandestine operations on the Kremlin's behalf. President Vladimir Putin has said the group does not represent the Russian state, but that private military contractors have the right to work anywhere in the world as long as they do not break Russian law.
The United Nations has repeatedly accused Malian soldiers of summarily executing civilians and suspected militants over the course of their decade-long fight against groups linked to al-Qaida and Islamic State.
The military has in some cases acknowledged that its forces were implicated in executions and other abuses, but few soldiers have faced criminal charges.
Meanwhile a surge in attacks since early March by an Islamic State affiliate has killed hundreds of civilians, according to official and military sources.