The Japanese subspecies of Hodgson's Hawk-eagle, Spizaetus nipalensis orientalis, is considered threatened and has been designated as Endangered by the Japanese government. We determined the complete mitochondrial DNA (mtDNA) sequence of this species and designed a primer set to amplify a highly variable region of mtDNA, part of the control region (CR), based on this complete sequence. Using the primers, we amplified the CR and then determined the haplotypes of 178 samples collected at different sites in Japan. A nested cladistic analysis indicated that gene flow within some clades was restricted. The inference key implied that isolation by distance had caused the restriction of gene flow. Moreover, the ranges of the clades in which restricted gene flow was detected overlapped with the ranges of other clades. These results suggest that there is no fragmental population of Hodgson's Hawk-eagle in Japan and that this species has dispersed within short distances, at least in some lineages. Genetic diversity was high in comparison with other species. Therefore, at least in terms of genetic diversity, the Japanese population of Hodgson's Hawk-eagle is probably not in a critical situation.