The B52 Stratofortress, which is capable of carrying nuclear weapons, briefly roared over the Osan Air Base, some 72 kilometres (45 miles) south of the inter-Korean border, the military and an eye-witness said.
It circled once over the airbase before heading home.
The aircraft are known to have taken part in joint annual US-South Korea military exercises that have enraged Pyongyang, but their flights over South Korea are rarely publicised.
The last time such a flight was made public was in 2013, after North Korea carried out its third nuclear test.
At that time, the US dispatched both a B52 and the more sophisticated B2 stealth bomber to South Korea in a show of military muscle against the North.
Pyongyang on Wednesday conducted its fourth nuclear test, angering the international community and raising tensions across the inter-Korean border.
AFP