Human-mediated hybridization poses a serious threat to the genetic integrity of threatened inter-fertile species or subspecies, such as the bontebok (Damaliscus pygargus pygargus) and blesbok (Damaliscus pygargus phillipsi) in South Africa. The identification of pure and admixed populations is key to sound biodiversity conservation management and practices. Where identification based on morphometric measurements are complex, molecular analysis can provide a tool to identify hybrids or introgression in populations. This study provides the first molecular analyses on pure bontebok and blesbok as well as putative hybrid populations and samples of unknown purity. Analysis of a panel of 13 microsatellite markers by applying a Bayesian model-based clustering programme provided evidence of clear partitioning between the two subspecies. Hybridization was detected in 33 % (40 of 121) of the samples with unknown purity. Currently, bontebok is listed as protected under the Convention on International Trade in Endangered Species of Wild Fauna and Flora and is listed as endangered under the United States of America’s Endangered Species Act. Our research found that the microsatellite markers used in this study could accurately identify pure and hybrid animals and can be used as a tool to conserve the genetic integrity of both subspecies.