The Japanese frog, Rana rugosa, has two distinct sex chromosome types, XX/XY and ZZ/ZW. These two types are found in localized groups, separated geographically by a boundary area predicted to lie somewhere around Lake Biwa in central Japan. To determine this precise boundary, the heterogametic sex of 18 populations around Lake Biwa was examined by genotyping sex-linked genes. Phylogenetic relationships between the populations were also analyzed using mitochondrial 12S rRNA gene. Results showed that the Suzuka-Kii mountain range located east of Lake Biwa separated the XX/XY populations from the ZZ/ZW populations. Unexpectedly, from a phylogenetic perspective, the ZZ/ZW populations around Lake Biwa belonged not to the main ZW group but to the XY group. The authors propose that the ZZ/ZW populations around Lake Biwa diverged secondarily from the XX/XY group through a change of heterogametic sex, eventually forming a new group. This group was thus named the 'Neo-ZW group'. As the main ZW group inhabiting northwestern Japan is known to have a different male heterogametic origin, this finding shows that change of heterogametic sex from male to female may have occurred twice, and independently, during the frog speciation.