Roads can fragment and isolate wildlife populations, which will eventually decrease genetic diversity within populations. Wildlife crossing structures may counteract these impacts, but most crossings are relatively new, and there is little evidence that they facilitate gene flow. We conducted a three-year research project in Banff National Park, Alberta, to evaluate the effectiveness of wildlife crossings to provide genetic connectivity. Our main objective was to determine how the Trans-Canada Highway and crossing structures along it affect gene flow in grizzly (Ursus arctos) and black bears (Ursus americanus). We compared genetic data generated from wildlife crossings with data collected from greater bear populations. We detected a genetic discontinuity at the highway in grizzly bears but not in black bears. We assigned grizzly bears that used crossings to populations north and south of the highway, providing evidence of bidirectional gene flow and genetic admixture. Parentage tests showed that 47% of black bears and 27% of grizzly bears that used crossings successfully bred, including multiple males and females of both species. Differentiating between dispersal and gene flow is difficult, but we documented gene flow by showing migration, reproduction and genetic admixture. We conclude that wildlife crossings allow sufficient gene flow to prevent genetic isolation.