Dozens of fire ants have been found at Hakata port in Fukuoka Prefecture, the Environment Ministry said, marking the invasive species' first appearance in Kyushu.
The stinging ants, native to South America, were first discovered in Japan in May, when they were spotted at a container yard at Kobe port. Several discoveries elsewhere on Honshu have since been made.
Along with the Hakata finding, announced Friday, fire ants have so far been found in Tokyo and five prefectures.
In Fukuoka, the ants were found in a crack in the port's container yard, which handles containers shipped from Nansha port in China's Guangdong province.
Authorities sprayed them with insecticide and deployed traps nearby, but no queen ants or larva were found.
The Environment Ministry suspects the ants arrived via containers from Nansha port that arrived on July 12, as no ants were found during a June 30 inspection.
The stings of the reddish-black fire ant are painful and can cause anaphylaxis in some people.