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Roads fragment ecosystems around the globe, but the effects of this fragmentation on biodiversity remain poorly understood. Wolverines (Gulo gulo) are snow-dependent carnivores that occur at low densities and they exhibit low genetic diversity at the southern extent of their range where they are snow-limited and fragmented by human development. Therefore, understanding the effect of roads and transportation infrastructure on population connectivity in protected strongholds such as national parks is crucial to effective wolverine management in a changing climate. We assessed whether the Trans-Canada Highway, Canada's largest east-west transportation corridor, affects wolverine gene flow in the Rocky Mountains. We used noninvasive genetic sampling methods (i.e., hair traps, backtracking) to collect DNA samples (i.e. hair, scat) from an 8000 km(2) area of Banff, Kootenay, and Yoho National Parks and provincial lands and then used population and individual-based genetic analyses (e.g., assignment tests, principal coordinates analysis) to examine genetic structure across the highway in the national parks complex. We collected 2586 DNA samples between 2010 and 2013 from which we identified 49 unique individuals (29 males, 20 females). We detected weak population structure in males and relatively strong genetic differentiation in females spanning the highway with complementary nuclear and mitochondrial DNA analyses. Our results demonstrate that sex-biased dispersal across a major highway can lead to genetic isolation and demographic fragmentation in a protected carnivore population, highlighting the urgent need to maintain connectivity for wildlife over an expanding global road network in the face of climate change, landscape degradation, and loss of biodiversity.