Objectives The Ainu, the indigenous people living on the northernmost island of Japan, Hokkaido, have long been a focus of anthropological interest because of their cultural, linguistic, and physical identity. A major problem with genetic studies on the Ainu is that the previously published data stemmed almost exclusively from only 51 modern‐day individuals living in Biratori Town, central Hokkaido. To clarify the actual genetic characteristics of the Ainu, individuals who are less influenced by mainland Japanese, who started large‐scale immigration into Hokkaido about 150 years ago, should be examined. Moreover, the samples should be collected from all over Hokkaido. Materials and methods Mitochondrial DNA haplogroups of 94 Ainu individuals from the Edo era were successfully determined by analyzing haplogroup‐defining polymorphisms in the hypervariable and coding regions. Thereafter, their frequencies were compared to those of other populations. Results Our findings indicate that the Ainu still retain the matrilineage of the Hokkaido Jomon people. However, the Siberian influence on this population is far greater than previously recognized. Moreover, the influence of mainland Japanese is evident, especially in the southwestern part of Hokkaido that is adjacent to Honshu, the main island of Japan. Discussion Our results suggest that the Ainu were formed from the Hokkaido Jomon people, but subsequently underwent considerable admixture with adjacent populations. The present study strongly recommends revision of the widely accepted dual‐structure model for the population history of the Japanese, in which the Ainu are assumed to be the direct descendants of the Jomon people.