Previous allozymic studies have revealed that Korean wild populations of Oryzias latipes have differentiated regionally, and are composed of two distinct groups, the East Korean Population and the China-West Korean Population. Recently, mitochondrial DNA (mtDNA) sequencing and restriction fragment length polymorphism (RFLP) analyses have confirmed these two groups, and shown that the distribution ranges of the two groups overlap in western Korea. In order to describe the detailed distributions of the two groups and the gene flow between them, genotypes of 13 allozymic loci were determined in 444 specimens from 96 localities in Korea. The two major groups were supported by remarkable allele frequency differences at six diagnostic loci: ACP*, AMY*, CK-A*, LDH-A*, PGM* and TF*. Individuals with the typical “eastern” genotype were mainly distributed in eastern and southern areas. In contrast, fish with the “western” genotype were predominant in the western area, and were further divided into two subgroups (the Han River and Geum River Subpopulations) by unique alleles at the ADH* locus. In the western coast, two distinct (eastern and western) genotypes were distributed in a mosaic fashion. This distribution pattern was identical to those from mtDNA analyses. Although the distribution patterns of the alleles at three loci (GPI-A*, LDH-C* and SOD*) showed introgressive conditions between the two groups, each population was nearly fixed as either the eastern or western genotype at all six diagnostic loci despite the proximity among samples. Therefore, it is suggested that some reproductive isolation mechanisms exist between the two groups in natural habitats.