The population genetic structure of Isaria cicadae, which caused enzootic of cicadas nymphs in three regions, was analyzed by ISSR marker. The results showed that all three enzootic populations showed high genetic diversity with the highest in the Jingtingshan population and the lowest in the Shitai population. The UPGMA clustering analysis revealed that different enzootic populations did not have a predominant lineage but were polyphyletic and heterogeneous. Genetic lineages had nothing to do with geographical origin. However, two subpopulations of Jingtingshan from different sampling periods were gathered into different clades, which exhibited remarkable temporal heterogeneity. The genetic differentiation (Gst) among populations (subpopulations) was 0.2153 and the gene flow was low at 0.9110 (Nm＜1), which indicated the low gene flow was one of the main reasons for the genetic variation in the population. Therefore, high heterogeneity and low dominance might be genetic structure characteristics of I. cicadae population causing enzootic of cicada nymphs.