A 224 bp fragment of the mitochondrial cytochrome b gene has been amplified from a 30-year-old mummy-like specimen of the Japanese river otter Lutra nippon by polymerase chain reaction (PCR). The amplified products were subcloned in the Smal site of pUC18 and sequenced. The sequence was different from those of the congeneric Eurasian otters Lutra lutra (Latvia) and Lutra lutra (China) in 7-9 nucleotides, all of which were located at the third position of a codon and identified as transitional differences A↔G or C↔T. The phylogenetic analysis using the 224 bp sequences of Lutra nippon, Lutra lutra (Latvia), Lutra lutra (China), Aonyx cinerea (Asian small-clawed otter), Mustela sibirica and Mustela itatsi (weasels) supports the recent morphological study that the Japanese river otter is not a subspecies of Lutra lutra, but a distinct species, Lutra nippon. We found that Lutra nippon and Lutra lutra contain the cytochrome b-like sequences, that appear to be a pseudo-form of cytochrome b gene. The sequences are characterized by the presence of deletion and termination codons, by the presence of several types of sequences with minor variations, and by the faster evolutionary rate compared with that of the mitochondrial cytochrome b gene, The genes would present in the nuclear DNA rather than in the mitochondrial DNA, as in the case of the nonfunctional cytochrome b-like sequences previously reported in a rodent.