Isozyme analysis, morphometric comparison, and artificial hybridization test wereperformed to elucidate the patterns of genic variation, morphological differentiation, genetic incompatibility, and a probable path in speciation between two MDH allelotypes (MM type and MS type) of the Dark Chub Zacco temmincki, a fresh water fish inhabiting in Korean waters. The degree of genic variation of MS type(HD=.023, HG=.021) was twofold higher than that of MM type (HD=.013, HG=.014) but both allelotypes were far less than the average genic variation of fresh water Bish in general. The average genetic similarities among 7 populations of MM type and 6 Populations of MS type were S=.947 and S=.966 respectively, whereas the value between two allelotypes was S=.853. Presumed divergent time of two allelotypes was estimated to be about 700 thousand years ago. Discriminant function analysis based on 18 morphometric characters of 302 specimens representing 12 populations revealed no morphological difference between two allelotypes. Artificial hybridization test indicates that there is an obvious genetic incogpatibility between two allelotypes and therefore it is assumed that isolating mechanism is completed.