While African leopard populations are considered to be continuous as demonstrated by their high genetic variation, the southernmost leopard population exists in the Eastern and Western Cape, South Africa, where anthropogenic activities may be affecting this population's structure. Little is known about the elusive, last free-roaming top predator in the region and this study is the first to report on leopard population structuring using nuclear DNA. By analyzing 14 microsatellite markers from 40 leopard tissue samples, we aimed to understand the populations' structure, genetic distance, and gene flow (Nm). Our results, based on spatially explicit analysis with Bayesian methods, indicate that leopards in the region exist in a fragmented population structure with lower than expected genetic diversity. Three population groups were identified, between which low to moderate levels of gene flow were observed (Nm 0.5 to 3.6). One subpopulation exhibited low genetic differentiation, suggesting a continuous population structure, while the remaining two appear to be less connected, with low emigration and immigration between these populations. Therefore, genetic barriers are present between the subpopulations, and while leopards in the study region may function as a metapopulation, anthropogenic activities threaten to decrease habitat and movement further. Our results indicate that the leopard population may become isolated within a few generations and suggest that management actions should aim to increase habitat connectivity and reduce human-carnivore conflict. Understanding genetic diversity and connectivity of populations has important conservation implications that can highlight management of priority populations to reverse the effects of human-caused extinctions.