Three people are reported to have been killed and many more injured in the latest quake, measuring magnitude 7.3, which came just a day after another tremor that claimed nine lives in the same area.
An apartment fire was blamed for at least one of the deaths, as the fresh quake sparked a fresh wave of destruction and injuries.
"We are also checking if any more people failed to escape," city official Kiichiro Terada said, adding that the fire was under control.
Local broadcasters showed images of toppled buildings and a bridge collapse in the hard-hit city of Kumamoto.
The US Geological Survey said the quake struck at 1:25am (local time) and had a relatively shallow depth of 10 kilometres.
The Japan Meteorological Agency initially issued a tsunami warning for the western coast of the Kyushu island, where hundreds were injured in Thursday's quake, but later lifted it.
Public broadcaster NHK showed footage of rescuers carrying a victim on a stretcher from a collapsed house to an ambulance while they massaged the person's chest.
NHK said authorities were considering evacuating patients from a Kumamoto city hospital over fears it could collapse as a wave of aftershocks shook the area.
The hospital was slanted and soldiers were sent to the scene to assess the situation, according to reports.
Scores of people were also reportedly trapped in a nursing home in Mashiki.
Local media showed images of rescue workers removing debris from atop a collapsed house in an apparent search for anyone trapped underneath.
Other footage showed residents gathering in a park and also scenes from the NHK bureau in Kumamoto city immediately after the quake, with objects falling off desks and shelves.
Hisako Ogata, 61, evacuated to a nearby park with her daughter, where some 50 other people sat on blue plastic sheets.
"We left my house as we could not stay due to continuous jolts," Ogata told AFP.
"It was so scary. Thank God, we are still alive."
Prime Minister Shinzo Abe said the Government was making every effort to determine the extent of the damage, carry out rescue and recovery, and to get accurate information to citizens.
Thursday's quake killed nine people and injured nearly 900.
Shotaro Sakamoto, a Kumamoto prefectural official, said the most recent quake was similar in terms of the shaking felt.
"The latest earthquake felt as strong as, or stronger than the original one," he said.
"It was really strong … Many people on the street appeared panicked."
Tokohu University professor Shinji Toda added: "Thursday's quake might have been a foreshock of this one."
Several aftershocks rattled the region later on Saturday, including two of nearly magnitude 6, and the Meteorological Agency warned of more.
"We are making every effort to respond to this quake," Chief Cabinet Secretary Yoshihide Suga said.
He confirmed there were no problems at any of the three nuclear power plants in the region.