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Lepus yarkandensis, an endemic hare species in the Tarim Basin of China, has been suffering from habitat fragmentation due to desert expansion. To evaluate the effect of habitat fragmentation on its genetic diversity, the genetic diversity based on male-specific SRY gene marker is examined. A relatively low level of SRY genetic diversity is found compared to previous studies with mtDNA data, possibly due to the low SRY mutation rate and positive selection. Furthermore, one haplotype exists in eight populations along the Tarim River but not in many other relatively isolated populations, suggesting that habitat fragmentation may affect population divergence. Despite this, our pairwise Fst analysis shows no significant differentiation among populations, and this may be mainly caused by positive selection on the SRY gene in that 88 percent of individuals share the same haplotype. Finally, the phylogenetic analysis shows deep differentiation between L. yarkandensis and other two hare species (L. capensis and L. europaeus).