Genetic relationships among 37 local populations of Geothelphusa dehaani from southern Kyushu, Japan, were examined by electrophoretic analysis at 15 loci encoding 12 enzymes and a general protein. Local populations from the mainland of Kagoshima Prefecture, southern Kyushu, were genetically interrelated, independent of body coloration (red, blue, or two-tone types). Populations from the islands neighboring the Kagoshima mainland were differentiated into two genetic groups, which are distantly related to each other, also independent of body coloration, except for the populations from the Koshikijima Islands, west of Kagoshima mainland. One group of island populations was genetically associated with the mainland populations, both forming a clade of a mainland lineage. The other group, recognized as an island lineage, was highly differentiated from the mainland lineage. Both lineages, mainland and island lineages corresponding to blue and red color types, respectively, were sympatrically distributed on Koshikijima Islands, thus possibly suggesting a secondary contact. It is suggested that the observed genetic differentiation among local populations of G. dehaani in southern Kyushu possibly resulted from geographic isolation, consistent with the past geological process of island formations in the region.