We examined allozymic variation in Hynobius boulengeri, H. stejnegeri, and an unidentified salamander from Amakusa Island, to elucidate the evolution of these salamanders within Kyushu, southern Japan. We found three genetic groups in Kyushu: (1) H. boulengeri from the Sobo-Katamuki Mountains, (2) H. stejnegeri from the Kyushu Central Mountains, and (3) H. boulengeri from the Osumi Peninsula and the salamander from Amakusa Island. Collectively, these three groups formed a clade that was genetically very remote from H. boulengeri from Shikoku and Honshu. The salamander from Amakusa Island was genetically very close to the southernmost populations of H. boulengeri from the Osumi Peninsula and was judged to be a population of the Kyushu group of H. boulengeri. Hynobius stejnegeri also proved to be genetically very close to the Kyushu populations of H. boulengeri, and was assumed to represent one lineage, which attained its unique amber body color in the course of differentiation from the ancestral Kyushu stock of H. boulengeri. The three genetic groups are surmised to have been isolated to their present ranges as a result of geohistoric events within Kyushu Island.