We surveyed geographic enzyme variation in Hynobius boulengeri by starch gel electrophoresis using a total of 181. salamanders belonging to 13 populations (10 populations of H. boulengeri and one population each of H. sp. from the Osumi Peninsula of Kyushu, H. naevius, and H, dunni for comparisons). Hynobius boulengeri exhibits substantial genetic differentiation, and can be divided into three groups (mainland Honshu, Shikoku, and Kyushu). Hynobius sp. from the Osumi Peninsula belongs to the Kyushu group of H. boulengeri. Genetic differentiation of the Honshu group and the other groups of H. boulengeri was great enough to separate them as distinct species. Closer relationships of the Shikoku and Kyushu groups conform to the patterns of differentiation and/or distribution recognized in other organisms occurring in these two regions, The pattern of geographic variation among populations of H. boulengeri seems to have been strongly affected by the geohistory of their distributional ranges.