The Przewalski's gazelle (Procapra przewalskii) is critically endangered and has experienced significant decline in population numbers in the past, and now survives in four isolated populations: the Bird Island, Hudong-Ketu, Yuanzhe and Shadao-Gahai populations. To understand better the effect of habitat fragmentation and geographical isolation on gene flow and genetic variation, and uncover genetic units for conservation, we examined the hypervariable region (420 bp) of the mitochondrial DNA control region of 29 Przewalski's gazelles from four localities distributed throughout their present geographical range. Phylogenetic analysis consistently revealed four distinct mtDNA lineages within Przewalski's gazelle, with each lineage restricted to a single geographical location. The study revealed a marked divergence of lineages from the Bird Island population relative to other populations, which corresponds to the geographical association. The nucleotide diversity within populations was very low (less than 0.004). The value of Nm was less than 0.3 individuals per generation, indicating low gene flow between populations. It was suggested that the Yuanzhe, Hudong-Ketu and Shadao-Gahai populations be managed as a conservation unit because of their genetic and geographical closeness.