The genetic variation in Eumeces latiscutatus from the main islands of Japan and the northern Ryukyus was investigated and compared with that of its close relatives (E. okadae and E. marginatus) using allozyme data. All three samples from the northern Tokara Island Group, currently identified as E. latiscutatus, were shown to belong to E. marginatus and not to E. latiscutatus. The non-monophyly of the northern Tokara samples and the great genetic differences within these samples may have resulted from colonization from more than one source population by northeastward overseas dispersal. The samples from the Izu Peninsula were genetically much closer to E. okadae than the other conspecific ones. This indicates that the samples from the Izu Peninsula and the other samples of E. latiscutatus should be treated as distinct species, and that E. latiscutatus from the Izu Peninsula and E. okadae from the Izu Island Group may be treated as conspecific. Samples from western Japan were genetically well differentiated from those of eastern Japan. Within the western group, the samples from the Osumi Island Group was genetically distinct from those from the other regions, by possessing unique alleles. Our phenograms also reveal a distant affinity between samples from the Danjo Island Group and the main islands of Japan. This may be the result of long geographic isolation of the Osumi and Danjo Island Groups from Kyushu. By contrast, samples from Sapporo and Aomori were poorly differentiated genetically in spite of the long separation of these two localities by the Tsugaru Strait. This suggests that overseas dispersal of E. latiscutatus occurred across this strait after its formation.