The relative contribution of Sepioteuthis sp. 1 and Sepioteuthis sp. 2 to oval squid fishery stocks in western Japan was examined using microsatellite DNA and partial sequences of mitochondrial cytochrome oxidase subunit I. A total of 1718 Sepioteuthis spp. individuals from 19 populations were identified to determine the proportion of each species in fishery stocks. Samples from Tanegashima and Yakushima Islands in southwestern Japan were also examined to determine depth distributions. DNA was used to refer 233 (17.3 %) individuals to Sepioteuthis sp. 1 and 1421 (82.7 %) individuals to Sepioteuthis sp. 2. Sepioteuthis sp. 1 was found on the Pacific side of mainland Japan, but was predominant in Tanegashima (77.1 %) and Yakushima Islands (100 %), suggesting its distribution was limited by the warmer waters of the Kuroshio Current. In contrast, Sepioteuthis sp. 2 was most commonly found around mainland Japan. Whereas Sepioteuthis sp.1 was caught in 10 to 53 m depth, Sepioteuthis sp. 2 was caught in 2 to 30 m depth. Differences in the spatial and bathymetric distributions of these two species might reflect differences in temperature preference, spawning depth, and migration patterns. Our results would be useful for managing the fishery stocks and spawning grounds of both species.