The level of genetic diversity and population differentiation of oval squid Shiro-ikaSepioteuthis sp. 2 around mainland Japan were examined using highly polymorphic DNA markers. Ten microsatellite loci showed relatively low genetic diversity (average observed heterozygosity HO = 0.68, overall mean number of alleles NA = 10.0) as compared with other squid, which may be explained by a recent history of population expansion. Populations from 12 sites along the coastline of Japan were genetically homogeneous, as measured by FST values (global FST: −0.0006; pairwise FST: from −0.0046 to 0.0083), RST values (global RST: 0.0025; pairwise RST: from −0.0076 to 0.03), a neighbor-joining (NJ) tree based on Cavalli-Sforza and Edwards chord distance, Bayesian clustering or analyses of three geographical groups (Sea of Japan, Seto Inland Sea, and Pacific Ocean; FCT = −0.0005), and should be considered as a single fishery stock. Allele frequencies also showed a high degree of similarity across different years in the samples taken from Hiroshima. Factors influencing this homogeneity include genetic exchange without barriers preventing gene flow, larval dispersal, and active adult migration of Sepioteuthis sp. 2.