The genetic diversity of Auricularia polytricha among 27 wild populations in China was analyzed by using inter-simple sequence repeat (ISSR) markers. At the species level, a total of 509 loci were amplified using eleven ISSR primers with the percentage of polymorphic loci (P) = 100%, Nei's gene diversity (H) = 0.1935, Shannon information index (I) = 0.3274, and total genetic diversity (H_t) = 0.1918. At the population level, P = 35.3%, H = 0.1277 and I = 0.1907 whereas diversity within populations (H_s) is 0.1135. A high degree of genetic differentiation (G_st = 0.4081) among populations was detected. This genetic structure is likely due to the combined effects of the limited gene flow (N_m = 0.7251), geographical isolation, and random genetic drift. Clustering analysis revealed that a remarkable geographical relatedness exists among strains within population, whereas no distinct geographical pattern occurs in the genetic variation among populations. Furthermore, clustering analysis did not suggest genetic variation to be correlated with the host association.