Abstract: Roughskin sculpin, Trachidermus fasciatus is a catadromous fish, and it is distributed along the coast of Northwest Pacific. The fish population has been greatly decreased in recent years due to degradation of habitats, water pollution and dam construction. In order to protect the fish population, it is necessary to study genetic structure of the fish population, which is very important for fishery management and conservation. Forty seven individuals used in this study were collected from 8 sampling locations in the coast of China and Japan, i.e., Hangzhou Bay (HZB), Wendeng (WD), Rongcheng (RC), Dongying (DY), Qinhuangdao (QHD), Dalian (DL), Dandong (DD) and Ariake Sea. The mitochondrial cytochrome b (Cytb) gene sequence of T. fasciatus was amplified by polymerase chain reaction (PCR) technology and sequenced on an automatic sequencer with both forward and reverse primers. The sequences were analyzed by using some software, which included Dnastar, Arlequin3.1, Mega4.0, Modeltest3.7 and Paup4.0. The results were as follows: the Cytb sequence of each specimen was 1141 bp and used for genetic diversity analysis. There was neither insertion nor deletion found among 38 mutations of nucleotide acids and 31 haplotypes were found among 47 individuals from 8 sampling sites. All 8 populations were characterized by high haplotype diversity (0.60-1.00) and low nucleotide diversity (0.0005-0.0041). The analysis result of molecular variance (AMOVA), median-networks and NJ phylogenetic trees showed that there was large genetic differentiation between those two regions, while there was no genetic variance among populations of the Chinese lineage. Net average genetic distance between two lineages was 0.82%. Applying divergence rate of Cytb gene sequence, the divergence of lineage China and Japan occurred about 410000 years ago. The mismatch distribution of pairwise nucleotide and the negatively selective neutrality test suggest that a recent population expansion had occurred in Chinese lineages, and the time was estimated to be about 120000 years before present (during the late Pleistocene). Two distinct lineages found in T. fasciatus were probably related to the Pleistocene coastal glaciations and the long geographical distance. Dispersal ability, coastal currents and the population expansion may be responsible for the homegeneity among the Chinese populations.