We investigated the genetic population structure of the Japanese grass lizard, Takydromus tachydromoides, based on polymerase chain reaction—restriction fragment length polymorphisms (PCR-RFLPs) and DNA sequence analyses of the mitochondrial cytochrome b gene. The PCR-RFLP analysis of 163 specimens collected from the main islands of Japan and the adjacent islands identified a total of 25 mitochondrial haplotypes (mitotypes). The phylogeny of the mitotypes revealed that Osumi and Tokara Island populations were remarkably diverged from all other populations on the main islands of Japan. Furthermore, several regional groups were also recognized at the western part of Japan, namely mitotype groups in Kyushu, those in the western Honshu, and those in the central Honshu and Shikoku. In contrast, little genetic diversity was observed throughout eastern Japan. The regional genetic differentiation and recent range expansion of this species are considered to be associated with past geological events and climate changes.