A total of 142 samples of bigfin reef squid (Sepioteuthis lessoniana FÉRUSSAC IN LESSON, 1831) were collected from the Andaman Sea, Gulf of Thailand, and South China Sea to study their morphological characteristics and assess their genetic divergence. Morphological variations were observed in their body size, number of sucker ring teeth, and the radula. Genetic sequences were obtained using cytochrome c oxidase subunit I (COI) gene and 16S ribosomal RNA (16S rRNA) gene markers. Two size variants (small and medium) were identified, which could be distinguished based on body size, number of sucker ring teeth, and characteristics of the radula. The medium-size form comprised 101 individuals, the small-size form comprised 39 individuals, and there were 2 intermediate individuals.In the genetic analysis, there were 2 distinct genetic lineages based on the 40 haplotypes discovered across the study areas. The small-size form group was dominant in Taiwan Strait, and the medium-size form group was widely dispersed and dominated elsewhere in the South China Sea and Andaman Sea. Results of the morphological analysis indicated a correlation with high level of genetic differentiation clearly separating the 2 size groups and revealing the existence of 2 sub-species of S. lessoniana in the study areas that could be recognized by the differences in their DNA sequences and morphometrics.