Pholiota microspora (“nameko” in Japanese) is one of the most common edible mushrooms, especially in Japan, where sawdust-based cultivation is the most dominant method accounting for 99% of the production. The current strains for sawdust cultivation in Japan are considered to have been derived from a single wild strain collected from Fukushima, Japan, implying that commercial nameko mushrooms are derived from a severe genetic bottleneck. We tested this single founder hypothesis by developing 14 microsatellite markers for P. microspora to evaluate the genetic diversity of 50 cultivars and 73 wild strains isolated from across Japan. Microsatellite analysis demonstrated that sawdust-cultivated strains from Japan were significantly less genetically diverse than the wild strains, and the former displayed a significant bottleneck signature. Analyzing the genetic relationships among all genotypes also revealed that the sawdust-cultivated samples clustered into one monophyletic subgroup. Moreover, the sawdust-cultivated samples in Japan were more closely related than full-sibs. These results were consistent with the single founder hypothesis that suggests that all commercial nameko mushrooms produced in Japan are descendants of a single ancestor. Therefore, we conclude that cultivated P. microspora originated from a single domestication event that substantially reduced the diversity of commercial nameko mushrooms in Japan.