Rhyzopertha dominica (F.) is a destructive insect pest of stored products. Due to this pest's broad host range, short life cycle, and high reproductive capacity, it has rapidly spread throughout the world. Understanding the population structuring of this important pest is vital for developing and implementing an effective integrated pest management strategy. In this study, we isolated 19 novel polymorphic microsatellite loci from an enriched genomic library based on a biotin/streptavidin capture protocol. The genetic parameters were estimated based on 80 individual R. dominica from two natural populations. The numbers of alleles ranged from 3 to 10, and sixteen loci exhibited polymorphic information contents (PICs) greater than 0.5. The observed (HO) and expected (HE) heterozygosities ranged from 0.033 to 0.900 and 0.310 to 0.882, respectively. Six locus/population combinations significantly deviated from Hardy–Weinberg equilibrium (HWE). These microsatellite markers will provide a valuable resource for studying the population genetic structure of this species.