Biological diversity is being lost at unprecedented rates, with genetic admixture and introgression presenting major threats to biodiversity. Our ability to accurately identify introgression is critical to manage species, obtain insights into evolutionary processes, and ultimately contribute to the Aichi Targets developed under the Convention on Biological Diversity. The current study concerns roan antelope, the second largest antelope in Africa. Despite their large size, these antelope are sensitive to habitat disturbance and interspecific competition, leading to the species being listed as Least Concern but with decreasing population trends, and as extinct over parts of its range. Molecular research identified the presence of two evolutionary significant units across their sub-Saharan range, corresponding to a West African lineage and a second larger group which includes animals from East, Central and Southern Africa. Within South Africa, one of the remaining bastions with increasing population sizes, there are a number of West African roan antelope populations on private farms, and concerns are that these animals hybridize with roan that naturally occur in the southern African region. We used a suite of 27 microsatellite markers to conduct admixture analysis. Our results indicate evidence of hybridization, with our developed tests using a simulated dataset being able to accurately identify F1, F2 and non-admixed individuals at threshold values of qi > 0.80 and qi > 0.85. However, further backcrosses were not always detectable with backcrossed-Western roan individuals (46.7-60%), backcrossed-East, Central and Southern African roan individuals (28.3-45%) and double backcrossed (83.3-98.3%) being incorrectly classified as non-admixed. Our study is the first to confirm ongoing hybridization in this within this iconic African antelope, and we provide recommendations for the future conservation and management of this species.