Ancient DNA was analyzed from skull remains of 12 brown bears (Ursus arctos) excavated from the archeological site of the Okhotsk Culture on Rebun Island of Hokkaido, where no natural populations of brown bears currently occur, in order to trace their original habitats. The Okhotsk Culture developed around southern coastal regions of the Okhotsk Sea (southern Sakhalin, Rebun and Rishiri Islands, northern and eastern Hokkaido, and southern Kuril Islands) during 6–11th centuries, A.D. The ancient people of those days are considered to have involved brown bears for traditional ceremonies and rituals. From the skull remains, partial fragments (approximately 250–360 base pairs) of the mitochondrial DNA (mtDNA) control region were successfully sequenced. Compared with sequence data of modern brown bears of the Hokkaido main land, ancient mtDNAs of Rebun Island were phylogenetically classified into either of two lineages of modern mtDNA: the north-central Hokkaido lineage and southern Hokkaido lineage. The southern Hokkaido lineage was identified from three juvenile (less than one year old) ancient bears, while the north-central Hokkaido lineage was mainly from adults (more than three years old). Our findings demonstrated that juvenile ancient bears of Rebun Island were originated from southern Hokkaido, which was an outside area of the Okhotsk Culture and belonged to the Epi-Jomon Culture with a close relation to a northern part of the Tohoku district. The molecular phylogeographic study on ancient and modern brown bears provides an insight to further understanding zooarcheology and ancient people's cultures around Hokkaido.