The airspace was declared as Air Defense Identification Zone (ADIZ) by China a few days ago, a development termed as provocative and dangerous by the US.
While the Pentagon did not confirm or deny the B-52 bombers, its spokesperson told reporters that two of its aircraft flew through the dispute zone of East China Sea, without informing China.
"Last night we conducted a training exercise that was long-planned. It involved two aircraft flying from Guam and returning to Guam," Pentagon spokesman Colonel Steven Warren, said adding that China was not informed about it.
There was no immediate reaction from China. The flight was part of a pre-scheduled exercise, he said.
"It continues to be our view that the policy announced by the Chinese over the weekend is unnecessarily inflammatory and has a destabilising impact on the region, when the fact of the matter is these are the kinds of differences that should not be addressed with threats or inflammatory language, but rather can and should be resolved diplomatically," White House Deputy Press Secretary Josh Earnest told reporters.
The State Department said the US is in consultation with its other friends and allies in the region including Japan and South Korea.
"This unilateral action appears to be an attempt to unilaterally change the status quo in the East China Sea. And this will raise regional tensions and increase the risk of miscalculation, confrontation and accidents. We have made this case to China," State Department spokesperson Jen Psaki, told reporters.
The US has raised the issue with China, she said. "We've urged the Chinese to exercise caution and restraint. We're also consulting with Japan and other affected parties throughout the region in response to this announcement," Psaki said.
She said the US has long talked about concerns about increasing tensions or the raising of tensions and the impacts that would have.
"At this point, our role is to continue to encourage both sides to move forward with dialogue, to express concerns when we disagree with steps that China has taken, which is the case we've obviously done here. But our position on the islands that this impacts, of course, has not changed," she said.
Expressing concern over the Chinese decision, the Pentagon spokesperson said the situation in East China Sea is no where near war and is not heading towards an armed conflict.
The rules China announced over the weekend stated that it has effectively demanded control over the airspace above a swathe of the East China Sea.