Genetic diversity and population structure of Japanese populations of the oval squid, Sepioteuthis cf. lessoniana, were compared with populations from Taiwan and Vietnam using nucleotide sequences of the mitochondrial DNA non-coding region 2. In total, 402 nucleotide sequences representing 242 individuals from Japanese waters (Ishikawa, Japan Sea coast of Honshu; Tokushima, eastern Shikoku; Nagasaki, western Kyushu; and Okinawajima and Ishigakijima Island, in the Ryukyu Archipelago) and the East and South China Seas (Keelung, northern Taiwan; Vietnam, Gulf of Tonkin) were examined. Among the 29 haplotypes recognized, haplotype no. 1 was shared by more than 75% of individuals from Japanese localities, whereas it was found in less than 13% of specimens from the East and South China Seas populations. Conversely, the East and South China Seas populations included more than 30% individuals with haplotype no. 2, whereas less than 10% of haplotype no. 2 individuals were from Japanese localities. The differences of haplotype and nucleotide diversities between pooled Japanese populations (0.2639, 0.23%) and the East and South China Seas populations (0.7900, 1.01%) indicate that S. cf. lessoniana from Japanese waters exhibits lower genetic diversity. An analysis of molecular variance between the Japanese populations and the East and South China Seas populations was highly significant. A minimum spanning tree of 29 haplotypes and an Unweighted Pair Group Method with Arithmetic mean (UPGMA) tree based on pairwise F_ST comparisons also supported the separation between Japanese and the East and South China Seas populations. We suggest that the Kuroshio Current physically limits gene flow and has thus caused the differences in genetic diversity among the populations examined.