Two species of the true frog genus Rana (R. japonica and R. ornativentris) are widely distributed on the main islands of Japan with some degree of habitat overlap. These species could be key indicators for environmental conditions and accurate identification is important. However, morphological characters in early stages of development are often insufficient to distinguish the two species, although their presence is often inferred from egg masses. Therefore, we developed a PCR-RFLP protocol for efficient and cost-effective identification of the species. We used a short partial 16S rRNA fragment (approx. 550 bp) and two diagnostic restriction enzymes (Spe I and Hph I) to identify species from DNA material. This approach allowed us to accurately assign all samples even without locality data. We also confirmed the performance of our method using another brown frog, R. t. tagoi.