The population genetic structure of the neon damselfish (Pomacentrus coelestis) in the northwestern Pacific Ocean was revealed by the hypervariable control region of the mitochondrial gene (343 bp). In total, 170 individuals were sampled from 8 localities distributed between Taiwan and Japan, and 71 haplotypes were obtained through sequence alignment. High haplotype diversity (h = 0.956 ± 0.008) with low nucleotide diversity (π = 0.010 ± 0.006) was observed, and the results of the mismatch distribution test suggested that a historical population expansion after a period of population bottleneck might have occurred among P. coelestis populations. Based on the results of the UPGMA tree and AMOVA (Φct = 0.193, P < 0.05) analyses, fish populations from eight localities could be divided into two groups: one includes populations from localities around mainland Japan, and the other includes those from Okinawa and southern Taiwan. A genetic break was found between populations from mainland Japan and Okinawa, and this break was congruent with the pattern of phenotypic variations documented in previous studies. This evidence supports the latitudinal variation of reproductive traits among P. coelestis populations likely being genetically based. It is suggested that the changes in sea level and sea surface temperatures during past glaciations might have resulted in population bottlenecks in P. coelestis and the modern populations in the northern West Pacific are likely the results of recolonization after such events. The Kuroshio Current acts not only as a vehicle for larval transport along its pathway (between populations in southern Taiwan and Okinawa) but also as a barrier for larval dispersal across the Kuroshio axis (between populations in mainland Japan and Okinawa).