The ectomycorrhizal (ECM) fungal communities associated with eucalyptus were investigated in Japan, to elucidate whether ECM fungi can establish in a stand of unfamiliar host species. In this study, the root-associated fungal communities of eucalyptus and native host species were investigated in two sites in Japan. By comparing the composition and the potential hosts and distribution of obtained ECM fungi between eucalyptus and native host species, we aimed to clarify whether host specificity can limit the establishment of native ECM fungi in a forests of exotic host species, and in consequent, limit the distribution expansion of these native fungi to the geographic area where only eucalyptus and other unfamiliar hosts exist.