Eurasian yellow perch (Perca fluviatilis) and American yellow perch (Perca flavescens) are known to be endemic species in Eurasia and North America, respectively. The presence of endemic species on each continent suggests their independent evolutionary history. However, because of the morphological similarity, distribution pattern, and only recent fossil record, their divergence time and speciation of the two Perca species has long been controversial. Here, from the comparison of the entire nucleotide sequences of cytochrome b gene, large genetic divergence between the two Perca species is observed although they are morphologically similar each other. Among 1,140 base pairs, interspecific nucleotide differences are found at 130 sites $(11.4\%)$. The differences varies with codon position, showing 22 sites in the first, 5 sites in the second, and 103 sites in the third codon position. Considering the types of nucleotide changes, transitional differences are much more than transversional differences and its ratio turned out to be 5.19. The estimated divergence time of the two Perca species indicates that they were separated each other approximately in the late Miocene period, which implies the long history of speciation. With comparison of the inferred amino acid sequences, strong structural and functional constraints which seem to be maintained by the highly conservative amino acid residues or protein regions, as found in other taxonomic groups of organisms, are also recognized in the cytochrome b of the fishes examined.