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Comparative landscape genetics studies provide insights on the impact of landscape elements on gene flow patterns of different species inhabiting the same geographic area. We investigated the population genetic structure of two sympatric ungulates, roe deer Capreolus capreolus and Northern chamois Rupicapra rupicapra, in a mountain area of the central Italian Alps (Trentino, northern Italy). A total of 122 chamois and 72 roe deer samples were genotyped by two species-specific panels of 11 polymorphic microsatellite loci and analyzed by aspatial and spatially explicit analyses. While the roe deer population resulted unstructured, a clear population structure was detected in chamois, with two main groups, one inhabiting the eastern and the other spread in the western part of the study area. Landscape genetics analysis confirmed these scenarios and revealed a different effect of landscape on gene flow. An IBD (Isolation-By-Distance) model best explained genetic variation in roe deer, while IBR (Isolation-By-Resistance) was found as the process underlying genetic variation patterns in chamois, suggesting arable lands, coniferous forests, watercourses, and main roads as potential barriers. Species distribution and landscape use might explain these results: roe deer mostly occupy valley floors relatively connected to each other, and their spatial behavior may promote gene flow across areas. On the other hand, chamois prefer higher elevations and their movements may be hindered by valleys, rivers, and road networks. This study highlights the different impacts of natural and anthropic landscape elements on gene flow in two sympatric species, resulting from their different ecological requirements.