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Bears are globally threatened by habitat fragmentation, particularly due to roads, which can act as dispersal barriers. We used noninvasive hair sampling to study the genetic diversity of Asiatic black bears (Ursus thibetanus) in the Dong Phayayen–Khao Yai forest complex, Thailand, during 2012–2015. We used 9 microsatellite loci to investigate black bears inhabiting 2 sides of the complex that have been separated by a highway for 60 years. We genetically identified 49 individuals (31 males and 18 females) in Khao Yai and 45 individuals (25 males and 20 females) in Dong Phayayen. The genetic diversity level of Dong Phayayen was similar to Khao Yai; low differentiation (FST = 0.035) and no strong evidence of inbreeding was indicated for both populations (FIS = 0.13 and 0.07, respectively). However, the pairwise FST between Khao Yai and Dong Phayayen was notably higher (FST = 0.035 and 0.038, respectively) than within Dong Phayayen (0.004), suggesting an observable effect of the highway on the populations, with a current migration rate of 1% between the 2 forests. Thus, the road apparently acts as a significant dispersal barrier for the black bear. This population is of conservation concern because the estimated effective population size (Ne = 178 bears) was below the recommended size for black bears. The relatively long generation time of Asiatic black bears could be the main factor contributing to the time lag between placement of the highway and changes in genetic diversity between the populations. Our simulations indicated a strong negative impact of the highway on the genetics of this population within the next 10 generations and an increased risk of extinction if there is continued isolation. Our data also suggest that an assisted migration program via habitat corridors could reduce the impact of this highway and promote the persistence of bears.