To investigate the extent of genetic differentiation among wild populations of the Pacific cod Gadus macrocephalus, we have examined genetic polymorphism at five locations within Korean waters [Boryeong in the West Sea (WC-BR); Jinhae Bay in the South Sea (SC-JH); Jumunjin (EC-JM), Jukbyeon (EC-JB), and Bangeojin (EC-BJ) off the eastern coast of Korea] using mitochondrial DNA (mtDNA) control region and microsatellite DNA (msDNA) markers. Nucleotide sequence analysis of 584 bp in the variable portion of the 5' end of the mtDNA control region revealed 27 variable nucleotide sites among 184 individuals, which defined eight, three, and 11 haplotypes in the western, southern, and eastern coast populations, respectively. The mtDNA analysis revealed a low variability but significant local differentiation among populations from these three areas within Korean waters. msDNA analysis also revealed moderate polymorphism in the wild populations, with a mean of 13.8-22.6 alleles per locus for the five msDNA markers and observed (and expected) heterozygosities of 0.755 (0.825) for the WC-BR, 0.793 (0.810) for the SC-JH, 0.920 (0.905) for the EC-BJ, 0.783 (0.865) for the EC-JB, and 0.804 (0.812) for the EC-JM populations. Analysis of msDNA loci indicated that Pacific cod sampled at the WC-BR, SC-JH, and EC-JB sites belong to genetically distinct populations. However, no significant difference was found between the Pacific cod population from SC-JH and that from EC-BJ. Consequently, three genetically distinct populations, namely, WC-BR, SC-JH and EC-BJ, and EC-JB, were identified using msDNA analysis. These results indicate that genetically distinct populations of Pacific cod are present in Korean coastal waters where spawning aggregations occur.