The Japanese wood mouse Apodemus speciosus eats large, hard-walled walnuts of Juglans ailanthifolia immediately after finding or after hoarding them. However, not all individuals can efficiently eat the nuts. In this study, to examine local variation in the ability to eat walnuts, feeding behavior was compared among nine wood mouse populations, four from mainland Honshu, where the walnut tree is distributed, and five from the Izu Islands (30–100 km south of Honshu), which lack the tree species. To avoid the effects of pre-capture experience with walnuts, mice from areas lacking the walnut trees were used for testing, even in the Honshu sites. Most mice from Honshu were able to eat walnuts after a 14-day training period, whereas most insular mice could not, with the exception of mice on Kouzushima Island. An analysis of the population genetic structures of these mice based on sequences of the mitochondrial control (D-loop) region revealed that the four insular populations are genetically distinct from the mainland populations, whereas the Kouzushima population remains genetically similar to the mainland populations. The relatively recent colonization of Kouzushima may explain why mice from this island were able to feed on walnuts despite the lack of walnut trees on the island. Thus, walnut-feeding ability appears to have some innate basis in the Japanese wood mouse, and this trait would be selected for in a walnut-available environment as it would better enable mice to survive during food shortages.