Phellinus noxius is a pathogenic fungus that causes brown root rot disease in a variety of tree species. This fungus is distributed in tropical and sub-tropical regions of Southeast and East Asia, Oceania, Australia, Central America and Africa. In Japan, it was first discovered on Ishigaki Island in Okinawa Prefecture in 1988; since then, it has been found on several of the Ryukyu Islands. Recently, this fungus was identified from the Ogasawara (Bonin) Islands, where it has killed trees, including rare endemic tree species. For effective control or quarantine methods, it is important to clarify whether the Japanese populations of P. noxius are indigenous to the area or if they have been introduced from other areas. We developed 20 microsatellite markers from genome assembly of P. noxius and genotyped 128 isolates from 12 of the Ryukyu Islands and 3 of the Ogasawara Islands. All isolates had unique genotypes, indicating that basidiospore infection is a primary dissemination method for the formation of new disease foci. Genetic structure analyses strongly supported genetic differentiation between the Ryukyu populations and the Ogasawara populations of P. noxius. High polymorphism of microsatellite loci suggests that Japanese populations are indigenous or were introduced a very long time ago. We discuss differences in invasion patterns between the Ryukyu Islands and the Ogasawara Islands.