The Ezo wolf (Canis lupus hattai Kishida, 1931 ) is an extinct subspecies that inhabited Hokkaido in Japan until the middle of the Meiji Period. Because there are very few preserved skeletons, no osteological and/or genetic analyses of the Ezo wolf have been conducted. In this study, 20 cranial and eight mandibular characters were measured on Ezo wolf skeletons, and mitochondrial DNA (mtDNA) was analyzed to assess genetic relationships between the Ezo wolf and other wolf lineages, including the Japanese wolf on Honshu. The morphological study showed that the Ezo wolf is larger than the Japanese wolf and similar in size to the grey wolf of the Asian and American Continents. MtDNA control sequences (751 bp) from two Ezo wolves were identical to those from the Canadian grey wolf. The morphological and genetic characters indicate that the ancestor of the Ezo wolf was genetically related to that of the grey wolf in Canada.