We compared the breeding season and genetic population structure of six sentinel crab (Macrophthalmus banzai) populations in southwestern Japan. Ovigerous females from Northern group populations (Wakayama, Kochi, and Tanega-shima) were observed from March to September, whereas ovigerous females from Southern group populations (Amami-oshima, Okinawa-jima, and Iriomote-jima) were observed from October to May. Genetic analysis using two markers corresponding to mitochondrial DNA encoding cytochrome oxidase subunit I (COI) and the large subunit of ribosomal RNA (16Sr-RNA) revealed a marked genetic difference between the Northern and Southern groups. A genetic boundary exists between the Tanega-shima and Amami-oshima populations, which is consistent with the interpopulation difference in the breeding season.