We sought to evaluate the genetic variability of largehead hairtail (Trichiurus japonicus) in the western North Pacific for the purpose of determining natural management units applicable to the fisheries regulation and ecological conservation. To achieve this, we analyzed two homologous segments in each fish's mitochondrial DNA using the AMOVA statistical model. In total, 180 DNA samples were collected from the continental shelves of Japan, the Yellow Sea (YS), the East China Seas (ECS), the Taiwan Strait (TS), and the South China Sea (SCS). In general, the haplotype diversity in the Cyt-b gene and D-loop region was high (>0.92), while the nucleotide diversity was moderate (<0.011). Our AMOVA model showed statistically significant genetic difference between the SCS and pan-ECS (Japan+YS+ECS+TS) populations. These subpopulations likely diverged as a result of the last geological sea-level recession event that created an outcrop within the Taiwan Strait, closing the waterway and gene flows between the ECS and SCS populations. Currently, sedentary life, limited dispersal ability, and modern monsoon-influenced marine flow patterns are the major forces that maintain the separation of these two hairtail populations. Our results support the creation of two independent management units for Trichiurus japonicus in the East China Sea and the South China Sea to protect the diversity of this species.