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Aim The aim of this study was to understand the roles of landscape features in shaping patterns of contemporary and historical genetic diversification among populations of the Andean tree frog ( Hypsiboas andinus) across spatial scales. Location Andes mountains, north-western Argentina, South America. Methods Mitochondrial DNA control region sequences were utilized to assess genetic differentiation among populations and calculate population pair-wise genetic distances. Three models of movement, namely traditional straight-line distance and two effective distances based on habitat classification, were examined to determine which of these explained the most variation in pair-wise population genetic differentiation. The two habitat classifications were based on digital vegetation and hydrology layers that were generated from a 90-m resolution digital elevation model (DEM) and known relationships between elevation and habitat. Mantel tests were conducted to test for correlations between geographic and genetic distance matrices and to estimate the percentage variation explained by each type of geographic distance. To investigate the location of possible barriers to gene flow, we used Monmonier’s maximum difference algorithm as implemented inbarrier 2.2. Results At both geographic scales, effective distances explained more variation in genetic differentiation than did straight-line distance. The least-cost distances based on the simple classification performed better than the more detailed habitat classification. We controlled for the effects of historical range fragmentation determined from previous nested clade analyses, and therefore evaluated the effect of different distances on the genetic variation attributable to more recent factors. Effective distances identified populations that were highly divergent as a result of isolation in unsuitable habitats. The proposed locations of barriers to gene flow identified using Monmonier’s maximum difference algorithm corresponded well with earlier analyses and supported findings from our partial Mantel tests. Main conclusions Our results indicate that landscape features have been important in both historical and contemporary genetic structuring of populations of H. andinus at both large and small spatial scales. A landscape genetic perspective offers novel insights not provided by traditional phylogeographic studies: (1) effective distances can better explain patterns of differentiation in populations, especially in heterogeneous landscapes where barriers to dispersal may be common; and (2) least-cost path analysis can help to identify corridors of movement between populations that are biologically more realistic. [ABSTRACT FROM AUTHOR]