In this study, 149 Himalayan marmot individuals from 13 populations were sampled from eastern Qinghai and genotyped at 11 microsatellite loci. Then the genetic diversity and genetic structure of these populations were analyzed using different population genetics methods. As the results showed, a total of 97 alleles were detected in all 11 SSR loci. The average observed heterozygosity and expected heterozygosity for each population ranged from 0.58 and 0.60 to 0.82 and 0.79, representing a relatively high level of genetic diversity. The genetic structure analyses demonstrated that the marmot populations in eastern Qinghai exhibited a strong genetic structure, where 13 geographical populations were allocated into 3 genetic clusters. Surprisingly, such three genetic clusters are in perfect accordance with the geographical units divided by the trunk stream of the Huangshui river and the head trunk stream of the Huanghe river. Therefore, we conclude that these two rivers act as natural barriers to the dispersal and gene-flow between the marmot populations in different units. Meanwhile, as suggested by the STRUCTURE analysis, there was still obvious gene-flow between the three genetic clusters, and the AMOVA analysis also showed that the percentage of the variance between genetic clusters was 6.60%, slightly higher than that between different populations within clusters (4.51%) and far lower than that within populations (88.90%), indicating a low degree of differentiation between the genetic clusters. All such results implied that the Himalayan marmots may cross over the rivers by some bridges or in low water periods to disperse and exchange. These results can provide scientific information for the monitoring and management of marmot populations and even plague control in this region.