Techniques for DNA extraction from small pieces of animal tissues and for polymerase chain reaction product-direct sequencing have been improved for molecular phylogenetic study. Using the techniques, the cytochrome b genes (375 bases) in the mitochondrial DNA were sequenced for three species of weasels distributed in Japan: Mustela itatsi, M. sibirica, and M. nivalis. M. itatsi and M. sibirica were identified by means of the ratio of the tail length to the head and body lengths. Five specimens of M. itatsi (a native Japanese species) showed 4.0-4.3% difference of the nucleotide sequence (15-16/375 bases) from two individuals of M. sibirica (a species introduced to Japan from Korea). A phylogenetic tree, constructed using sequence data, suggests that M. itatsi is distinct from M. sibirica in agreement with its morphological characters. M. nivalis was more distinct with a 6.4-6.9% sequence difference from the other two species. On the basis of nucleotide substitutions, the divergences of M. itatsi from M. sibirica and M. nivalis are estimated to have occurred around 1.6-1.7 million years ago and 2.7-2.8 million years ago, respectively. Thus, cytochrome b sequence analysis with the present improved methods provides a new clue for the development of the phylogenetic study of closely related mammalian species.