Pleuronectes yokohama and P. herzensteini, belonged to the family Pleuronectidae, are commercially important fisheries resources in Northwest Pacific Ocean. To date, no researches were carried out in the differentiation between the two species. Partial nucleotide sequences of mitochondrial DNA COI, Cyt b genes and D-loop were sequenced and analyzed for P. yokohama and P. herzensteini in the present day. 107 nucleotide substitutions were checked between the two species in the total 1 373 bp sequences analyzed, and most of them were synonymous transitions at the third codon positions in the two proteincoding genes. Nucleotide composition analysis indicated a strong bias against guanine (G) in the three fragments, especially in the third codon positions of COI and Cyt b gene. There was obvious difference on the degree of genetic divergence between the two species in the three mtDNA segments. The substitution rate for D-loop was the highest and the rates for COI and Cyt b were generally similar. It is suggested that gene markers used in future research should be selected carefully according to the different situations. The estimated divergence time between the two Pleuronectes species based on Cyt b gene was about 2 million years, which indicated a Pleistocene divergence for the two species.