The Tsugaru Strait is often referred to as Blakiston's Line in biogeography. Although this strait is thought to have become narrower during the Last Glacial Maximum, it did not close completely to form a land bridge. Sea‐level drawdown associated with cooling nevertheless weakened the influence of the Tsugaru Strait as a barrier to faunal migration. The currently accepted hypothesis is that only some large and highly mobile ungulates were able to migrate to Honshu over this strait to the exclusion of small mammals. However, this remains problematic as very few fossils corroborate this idea, and quantitative identification methods for use with tiny fragments of teeth have not been developed. In this study, we determine which of the species Lepus timidus (now distributed on Hokkaido and to the north) and Lepus brachyurus (now mainly distributed on Honshu, Shikoku, and Kyushu) can accurately be identified within a sample of Late Pleistocene Lepus sp. fossils recently unearthed from the Shitsukari‐Abe cave to the immediate south of the Tsugaru Strait. This is because we are unable to exclude the possibility that these fossils are referable to L. timidus on the basis of site location alone. Thus, utilising images of mandibular third premolar teeth obtained via microcomputed tomography scanning, we report (a) linear measurements, (b) a survey of the frequency of appearance of nonmetric traits, and (c) principal component analysis using landmark methods. The results show that fossil representatives of the genus Lepus can be differentiated from extant L. timidus and closely resemble L. brachyurus. Considering the past allopatric distribution of the 2 species suggested by other studies as well as fossil records around the Tsugaru Strait, this result corroborates the existing hypothesis that only large and highly mobile ungulates were able to navigate the Tsugaru Strait.