The bombers took off from the US Pacific territory of Guam on Tuesday night, before entering South Korean airspace and conducting firing exercises over the East Sea and Yellow Sea, South Korea's military said.
The two bombers flew to the Sea of Japan from Andersen Air Force Base in Guam.
"Participating in bilateral training enables the operational units to improve their combined capabilities and tactical skills, while also building bilateral confidence and strong working relationships", according to the statement. "This is a clear demonstration of our ability to conduct seamless operations with all of our allies anytime anywhere".
The latest in a series of simulated U.S. bombing runs around North Korea, the operation is described as a "show of force" amid ongoing tensions with North Korea.
An unusually aggressive approach to the North by Trump, which has included rhetoric hinting at U.S. strikes and threatening the destruction of North Korea's leadership, has some South Koreans fearful that war is closer than at any time since the Korean War ended in a shaky ceasefire in 1953.More news: Chicago Rethinks Soda Tax
Late last month, the United States flew B-1Bs over worldwide waters close to the North's east coast.
President Trump hosted a discussion on options to respond to any North Korean aggression, or, if necessary, to prevent Pyongyang from threatening the USA and its allies with nuclear weapons, the White House said in a statement.
The B-1Bs also conducted exercises with Japan's Air Force in what the US statement called a "sequenced bilateral mission".
Meanwhile, China - North Korea's closest ally and trading partner - expressed alarm at how far the rhetoric on both sides had gone, warning of the increased risk of a "fatal misjudgement"."The global community won't accept North Korea as a nuclear power". "These actions are consistent with long-standing and well-known US freedom of navigation policies that are applied to military operations around the world".