Images broadcast on local media showed the bus flipped on its side in the eastern city of Chattanooga with a tree splicing its center.
There were conflicting reports about the number of children killed, with multiple US media stating that six students had died, citing the Hamilton County Office of the District Attorney General. When contacted by AFP, the office said it had not released that figure.
"There were we believe 35 children on that school bus ranging from kindergarten through fifth grade. It was a single-vehicle crash," Chattanooga Police Chief Fred Fletcher told reporters.
The city's police department posted on Twitter that "multiple fatalities" had occurred, adding that the exact number was not confirmed.
"Certainly speed is being investigated very, very strongly as a factor in this crash," Fletcher said.
"It was clear and dry... There did not appear to be any roadway conditions. But it is a complicated ... Scene, it covers a significant area," he added.
The National Transportation Safety Board said on Twitter it would deploy a team today morning to investigate the crash.
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