Gansu zokors (Myospalax cansus) are small, solitary, subterranean rodents that inhabit the Loess Plateau in China. The genetic diversity and population genetic structure of M. cansus were determined by analyzing the sequence variation of a 530 bp fragment of the mitochondrial D-loop region in 158 natural individuals from eight locations in eastern Qinghai. Overall, 26 polymorphic sites were identified and 39 haplotypes were defined. Genetic diversity estimates revealed extensive haplotype diversity (0.9532) and limited nucleotide diversities (0.00636) in all populations. Analysis of neutral evolution and mismatch distribution implied that M. cansus might have experienced a recent population expansion. The median-joining network was star-like throughout the studied range of M. cansus and showed that most individuals from different populations were highly interconnected with each other and did not exhibit reciprocal monophyly. Population differentiation among populations was confirmed by the percentage of variation among populations and within populations, which were 51.18% and 48.82%, respectively. Nm analyses indicated that the gene flow was limited among sampled populations. A Mantel test revealed significant positive correlation between genetic distances, expressed by FST, and geographical distances among populations, excluding the PA1 population, suggesting that distance isolation plays a remarkable role in genetic differentiation. In the present study, the available data suggested that the Quaternary climatic oscillation and the weak dispersal ability of subterranean animals have shaped the peculiar population genetic diversity and genetic structure of Gansu zokors.