Astragalus mongholicus (Fabaceae) is a perennial herb and a widely used medicinal plant in traditional Chinese medicine (TCM) known as Radix Astragali (Huangqi). It was reported to have hepatoprotective, cardioprotective, antidiabetic, antiaging, sedative and immunopotentiating effects, and could also be used as an adjuvant medicine during cancer therapy. Until now, there is only a little research on its population genetics and no report on development of microsatellite loci for this plant. In this study, a microsatellite-enriched genomic DNA library of A. mongholicus was developed and screened to identify marker loci. Ten polymorphic loci were isolated and analyzed by screening 30 individuals. The number of alleles per locus ranged from 4 to 19, with an average of 12.3 alleles per locus. The observed heterozygosity (H_O) and the expected heterozygosity (H_E) ranged from 0.367 to 1.000 and from 0.395 to 0.912, respectively. The polymorphism information content (PIC) varied from 0.361 to 0.888, with an average of 0.762. This is the first report on characterization of microsatellite loci for A. mongholicus, and these markers will be useful for population genetics and molecular ecology studies of this plant.