Black muntjac are currently threatened by habitat loss, fragmentation and human hunting, which has led to the extinction of three subspecies in the wild. In order to design effective conservation strategies for the black muntjac, we have investigated genetic diversity and gene flow in the black muntjac populations by analyzing about 480 base pairs of the mitochondrial DNA (mtDNA) control region in 36 individuals sampled from Suichang, Songyang and Longquan. Black muntjac exhibited high mtDNA diversity with thirteen variable sites and twelve haplotypes identified. Neither the estimate of Tajima′s D nor that of Fu and Li′s D deviated significantly from the neutral selection hypothesis (P＞ 0.1) for three populations, showing no evidence of strong selective sweeps or balancing selection. Although there appears to be reasonably high gene flow among the three populations, these data single out the Suichang population as being highly genetically distinct and worthy of separate conservation consideration. Therefore, it is recommended that a breeding program for the Suichang population be established.