Lepidoglyphus destructor (Schrank) is a prominent and world-wide pest of stored grain. Currently, genetic diversity of L. destructor is still little known due to the limited number of studies. The objective of this investigation was to assess genetic diversity and differentiation of four populations of L. destructor from four geographic locations in China using inter-simple sequence repeat (ISSR) fingerprinting. A total of 58 DNA bands were tested, 47 of which were polymorphic and the percentage of polymorphic bands (PPB) was 81.03%. Nei's gene diversity (He) and Shannon's Information index (I) revealed that the genetic diversity was high. Further, the value of Gst was 0.5680, indicating a median level of genetic differentiation in these populations. At the same time, analysis of molecular variation (AMOVA) showed that 45.93% of variation can be identified within populations. The level of gene flow (Nm) was moderate. Cluster analysis suggested that genotypes isolated from the same locations displayed a higher genetic similarity than those from different ones, forming a specific clade according to their geographic locations. Nevertheless, there was no significant correlation between the genetic distance and geographic distance.