The Japanese frog Glandirana rugosa is unique in that it shows geographic variation in sex chromosome differentiation and heterogametic sex determination. To elucidate the cause of interpopulation differences in gonadal sex differentiation, we investigated hormonally induced sex reversal and the expression patterns of genes associated with sex determination during early tadpole development. We found that sex reversal was easily induced in XX females and XY males of 2 forms (West-Japan and East-Japan) of G. rugosa with the ancestral homomorphic sex chromosomes under male heterogametic sex determination. During sex reversal, expression of CYP19 and/or FOXL2 was dependent on the phenotypic sex of the gonad. In contrast, sex reversal was not induced in ZW females of a population with a heteromorphic ZW sex chromosome system or in XX females or XY males in a population with a heteromorphic XY sex chromosome system. The latter 2 populations are evolutionarily derived forms. These results indicate an evolutionary direction for the gonadal sex differentiation mechanism. The original system was highly sensitive to sex hormones and allowed almost complete sex reversal. From this ancestral form, a new system evolved that was resistant to hormones and showed a change in the heterogametic sex and the sex chromosome differentiation mechanism.