Mitochondrial DNA (mtDNA) D-loop control region sequences ranging in length from 583 to 598 bp were determined for eight Japanese wolf specimens (Canis lupus hodophilax Temminck, 1839) collected from several sites and compared with 105 haplotypes from the domestic dog (C. lupus familiaris) and continental grey wolf (C. lupus lupus). Also, a 197-bp mtDNA sequence was amplified from archaeological wolf specimens and two continental wolf specimens (C. lupus chanco) as reference sequences for analysis. The mtDNA haplotypes from the eight Japanese wolf specimens were closely related to each other and grouped in a single lineage with an 88% bootstrap value in a neighbor-joining analysis. The results provide valuable information for understanding the taxonomic and phylogenetic relationships of the Japanese wolf, which have long been controversial.