The taxonomic status of Trichiurus japonicus Temminck and Schlegel, 1844 as a valid species is still controversial, although it has long been considered to morphometrically and meristically differ from T. lepturus Linnaeus, 1758. A portion of the mitochondrial DNA encoding the 16S ribosomal RNA (16S rRNA) gene sequence was compared between T. japonicus (obtained from various parts of Japan) and T. lepturus, obtained from the Western Atlantic (around the type locality of T. lepturus) and Indo-Pacific waters. The intraspecific sequence divergences of the partial 16S rRNA gene (at ~600 bp) were calculated to be 0.6% - 1.0% and 0.2% -3.0% for T. japonicus and T. lepturus, respectively, while the interspecific divergence was much greater at 4.8% -7.0%. Phylogenetic analysis using a Neighbor-joining algorithm showed that the haplotypes of T. japonicus formed a separate cluster from both the Western Atlantic and Indo-Pacific populations of T. lepturus with no overlap or sharing between them. Furthermore, restriction digestion of unpurified PCR products with the Hinf I restriction enzyme generated reproducible species-specific restriction patterns showing 2 fragments (of 378 and 222 bp) in T. lepturus whereas no cleavage was observed in T. japonicus. This phylogenetic analysis study coupled with PCR- RFLP analysis confirms the validity of T. japonicus as a separate species (in accordance with previous morphometric classification) and rules out any synonymy with T. lepturus.