The long-tailed hamster Cricetulus longicaudatus is a dominant rodent in farmland of Shanxi Province, China, but little is known about its genetic diversity and population structure. In this study, the genomic DNAs of individuals from 13 populations captured in different fields of Shanxi were extracted and amplified by six pairs of microsatellite primers and by universal primers for mtDNA COI gene sequences. Our data revealed significant departure from Hardy-Weinberg equilibrium in four of the 13 populations. In all 13 populations, the mean observed heterozygosity was significantly lower than the expected heterozygosity. Meanwhile, the mean inbreeding coefficient was statistically significant, which indicated non-random mating within populations. The pairwise genetic distance and natural logarithm of linear geographical distance were not significantly correlated for any C. longicaudatus populations. However, the correlation between genetic distance and resistance distance based on mountain landscape was significant, suggesting that the mountain landscape is an important factor affecting gene flow of C. longicaudatus. Pairwise FST analysis of population structure showed moderate to high genetic differentiation among populations, and all individuals could be divided into two gene clusters. Phylogenetic analysis based on COI sequences also showed that many individuals originated from a single haplotype, suggesting the existence of gene exchange among these populations at some time in the past. Our research should provide a scientific basis for the analysis of genetic differentiation and gene flow among populations of C. longicaudatus.