Host specificity may potentially limit the distribution expansion of ectomycorrhizal (ECM) fungi into areas where their original host plants are absent. To test this hypothesis, we investigated whether populations of native ECM fungi may establish in stands of exotic host trees, namely those of the Eucalyptus species, in Japan. ECM fungal communities associated with eucalyptus and surrounding native host species (Pinus thunbergii and Fagaceae spp.) were investigated at two sites; one site in which eucalyptus and native trees were growing in isolation, and a second site in which these species were mixed. To identify fungal taxa, the nuclear ribosomal internal transcribed spacer region 1 was sequenced for the ECM fungi from the root tips and clustered into operational taxonomic units (OTUs). To confirm whether the retrieved OTUs were native to Japan, they were queried against the entire database of the National Center for Biotechnology Information, UNITE, and GlobalFungi, whereby sampling locations and associated hosts were obtained from sequences with ≥97% similarity. Eucalyptus trees were associated with seven and 12 ECM fungal OTUs, including putatively exotic OTUs in isolated and mixed sites, respectively. Among the 36 and 63 native ECM fungal OTUs detected from native hosts at isolated and mixed sites, only one OTU was shared with eucalyptus at the respective sites. This means that most native ECM fungi in Japan may be incapable of forming an association with exotic Eucalyptus spp. Notably, even ECM fungi associated with both Pinus and Quercus were not detected from eucalyptus, suggesting that host-fungus incompatibility is determined not only by host phylogenetic relatedness but also by host biogeographic affinities. Our findings show that the incompatibility with eucalyptus as well as dispersal limitation may prevent the distribution expansion of native ECM fungi in Japan into the distribution ranges of Eucalyptus spp., where the original hosts are absent.