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Aim Understanding how heterogeneous landscapes shape genetic structure not only sheds light on processes involved in population divergence and speciation, but can also guide management strategies to promote and maintain genetic connectivity of populations of endangered species. This study aimed to (1) identify barriers and corridors for gene flow among populations of the endangered frog, Atelopus varius and (2) assess the relative contributions of alternative landscape factors to patterns of genetic variation among these populations in a hypothesis testing framework. Location This study took place in western Panama and included all nine of the remaining known populations of A. varius at the time of study. Methods The influence of landscape variables on gene flow among populations was examined by testing for correlations between alternative landscape-resistance scenarios and genetic distance. Fifteen alternative hypotheses about the influence of (1) riparian habitat corridors, (2) steep slopes, and (3) climatic suitability on patterns of genetic structure were tested in a causal modelling framework, using Mantel and partial-Mantel tests, along with an analysis of molecular variation. Results Only the hypothesis attributing resistance to dispersal across steep slopes (genetic isolation by slope distance) was fully supported by the causal modelling approach. However, the analysis of molecular variance and the paths of least-slope among populations suggest that riparian habitat connectivity may influence genetic structure as well. Main conclusions These results suggest that patterns of genetic variation among A. varius populations are affected by the slope of the landscape such that areas with steep slopes act as barriers to gene flow. In contrast, areas of low slope, such as streams and mountain ridges, appear to be important corridors for gene flow, especially among high elevation populations. These results engender important considerations for the management of this critically endangered species. [ABSTRACT FROM AUTHOR]