The phylogenetic relationship, biogeography, and evolutionary history of closely related two firefly species,Curtos costipennisandC. okinawanus, distributed in the Ryukyu Islands of Japan were examined based on nucleotide sequences of mitochondrial (2.2 kb long) and nuclear (1.1-1.2 kb long) DNAs. In these analyses, individuals were divided among three genetically distinct local groups,C. costipennisin the Amami region,C. okinawanusin the Okinawa region, andC. costipennisin the Sakishima region. Their mtDNA sequences suggested that ancestralC. costipennispopulation was first separated between the Central and Southern Ryukyu areas, and the northern half was then subdivided betweenC. costipennisin the Amami andC. okinawanusin the Okinawa. The application of the molecular evolutionary clocks of coleopteran insects indicated that their vicariance occurred 1.0–1.4 million years ago, suggesting the influence of submergence and subdivision of a paleopeninsula extending between the Ryukyu Islands and continental China through Taiwan in the early Pleistocene.