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Population fragmentation is stressing wildlife species worldwide. In populations with minimal genetic structure across potential fractures, detecting fragmentation can be challenging. Here we apply a relatively unused approach, genetic pedigree analysis, to detect fragmentation in the American black bear (Ursus americanus) across 2 highway corridors that are bordered by large, contiguous populations. We compared our results with movements detected through Global Positioning System (GPS) telemetry of collared bears between 2005 and 2010. We used 20-locus microsatellite genotypes to identify 104 first-order relatives (parent–offspring or full siblings) within 383 black bears, sampled between 2002 and 2012. We compared numbers of pairs of immediate relatives found on either side of 2 highways—U.S. Highway 2 in northwestern Montana, USA, and BC Highway 3 in southeastern British Columbia, Canada—with an expected rate, the mean across 22 lines parallel to each highway at 1-km intervals. We found that over similar geographic scales, dispersal was lower across the transportation corridors than adjacent areas without a highway corridor. The observed number of migrants across Highway 2 was 3, well below the confidence interval of the expected number of 15.1 migrants/available bears (95% CI = 12.2–18.0). Highway 3 had 6 migrants, compared with the expected 13.1 bears (95% CI = 10.8–15.5). None of 16 black bears wearing GPS radiocollars for 1 year crossed Highway 2, yet 6 of 18 crossed Highway 3. These results suggest that even though 33% of radiocollared black bears crossed Highway 3, there appeared to be less dispersal across the transportation corridors than across other regions in the study area. Pedigree and telemetry results were more closely aligned in the Highway 2 system, with both methods suggesting more intense fragmentation than we found along Highway 3. Our results identified pedigree analysis as another tool for investigating population fragmentation, particularly in situations where genetic differentiation is too weak to determine migration rates using individual-based methods, such as population assignment.