Numerous studies have recognized a biogeographic boundary (i.e., Blakiston's Line) between Hokkaido and Honshu, Japan, where the Tsugaru Strait probably functions as a geographic barrier to the movement of terrestrial species. To clarify if this biogeographic boundary applies to soil microbes, we examined community structures of ectomycorrhizal (EM) fungi across the Tsugaru Strait. We sampled 266 soil cores from six conifer-dominated forests, and collected EM roots for fungal species identification based on internal transcribed spacer sequences. We found that EM fungal communities were primarily influenced by contemporary climate, but not significantly by the geographic barrier or spatial distance. Fungal OTUs appeared to be widespread in the Holarctic regions, suggesting long-distance dispersal capabilities or ancient migrations before the separation of Japanese islands from the continent. The significant effects of climate in this and previous studies imply that EM fungal communities may be sensitive to future climate change.