We investigated the population genetics and fine-scale genetic structure of Rhizopogon roseolus. A total of 173 R. roseolus sporocarps were collected from two stands in the Tottori sand dune. We developed and applied five novel polymorphic microsatellite (SSR; simple sequence repeat) markers for sporocarp genotyping. In total, we identified 110 genets, most of which were small in size. Spatial autocorrelation analyses revealed a significantly positive genetic structure in short-distance classes. The inbreeding coefficient value was significant in both stands (F_IS = 0.18), while the F_ST value (F_ST = 0.020) indicated little genetic differentiation between the two populations. The majority of alleles were distributed in both stands with similar frequencies. These results suggest that short-distance spore dispersal plays a dominant role in generating new genets, and eventually increases the frequency of inbreeding in the Tottori sand dune, whereas rare gene flow between the two stands, possibly associated with spore dispersal by mycophagous animals, could reduce genetic differentiation.