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Reduced gene exchange between animal populations may be an indicator of the effects of anthropogenic fragmentation or it may reflect natural gradients in the landscape that can also result in population fragmentation. It can be difficult, therefore, to disentangle the role of local ecology from anthropogenic factors, creating a risk of attributing a lack of gene flow as being due to human activities, leading to ill‐informed management decisions. Here, we test the ecological and anthropogenic factors driving population differentiation and show how the relative influence of such effects can be identified. Using Bayesian clustering and a causal modelling approach, we combine genetic and remote sensing data to disentangle the confounding influences of ecological and anthropogenic fragmentation. We investigate a region where such confusion may arise; in and around the Okavango Delta in northern Botswana. Specifically, we used 20 microsatellites to investigate the genetic structuring of African lions Panthera leo occupying a landscape dominated by two very different environments, the wetland Okavango and the surrounding Kalahari Desert. We find that differences in ecology, rather than anthropogenic barriers, are driving genetic differences in the population and that despite their ability to disperse long distances these lion populations are differentiated into two distinct genetic groups, one inhabiting the wetland Okavango Delta and the other one inhabiting the surrounding dryland Kalahari, divided by an apparently unobstructed boundary. The genetic structure observed could easily have been misinterpreted as a response to anthropogenic disturbance reducing gene flow. This reinforces the need to consider non‐anthropogenic hypotheses, such as ecological differences between habitats, when assessing possible mechanisms of gene flow and their implications for population management. As anthropogenic pressure increases in this region, we recommend conservation managers consider the Okavango population as a separate conservation unit, but also recognize the importance of maintaining the current structural landscape connectivity. [ABSTRACT FROM AUTHOR]