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The genetic diversity of Gasterophilus pecorum populations consisting of 192 individuals sampled from Przewalski’s horses (Equus ferus przewalskii) in Xinjiang Province, China, was evaluated using 12 microsatellite loci. The genetic variability within populations and genetic differentiation among populations were estimated. A total of 163 alleles were detected and the average value of observed number of alleles at each locus ranged from 7 to 19 (average 13.5625). The expected heterozygosity (He) varied from 0.5933 (GP361) to 0.9208 (GP253) and averaged 0.8426. The effective number of alleles (Ne) of the simple sequence repeat (SSR) markers was 7.1756, and it ranged from 2.4430 to 11.5214. The polymorphism information content (PIC) ranged from 0.5643 (GP361) to 0.9053 (GP253) (average 0.8119). The average Shannon’s information index (I) was 2.1272. Parameters of genetic diversity (Ht, Hs and Gst) and F-statistic (Fis, Fit and Fst) were estimated. The mean of total gene diversity (Ht = 0.8798) and the average interspecific genetic diversity (Dst = 0.0110) showed that G. pecorum had a relatively high level of genetic variation, which was mainly within populations. The Fixation indices as Fst ranged from 0.0029 to 0.0950, and the Number of per generation migrants (Nm) among the populations varied from 2.3814 to 85.9745. These results indicate that the heterozygosity and genetic diversity of the G. pecorum populations were very high and that gene exchange was frequent. Correlation analyses demonstrated that the number of alleles and genetic diversity of the populations were not significantly different (p = 0.2). Unweighted Pair Group Method with Arithmetic mean (UPGMA) clustering analyses revealed a genetic similarity of 0.0671 – 0.1118. Analysis of molecular variance (AMOVA) demonstrated that the percentage of variation in the intrapopulation was small (0.67%) and the genetic variation in the G. pecorum populations was mainly exist within the species. An understanding of population genetic structure is not only crucial for assessing the actions and interactions of evolutionary forces in natural populations but also helps elucidate parasitic strategies in G. pecorum.