Human–bear conflict is a significant and pressing social issue in Japan. Understanding genetic diversity, population structure, and local population size of the Asian black bear (Ursus thibetanus) is crucial not only for improving ecological and genetic knowledge of the species but also for developing effective conservation and management measures. We therefore examined sex, mitochondrial (mt) DNA variations, and 12 nuclear microsatellite genotypes from hair-derived DNA collected using 35 traps in the Kiso region, Nagano Prefecture. After rigorous verification of microsatellite genotyping, we identified 30 distinct individuals (including seven males and 18 females) within an approximately 48 km² area. MtDNA analysis revealed that all the males shared haplotype E01, while the females possessed three haplotypes, including E07, which was detected in Nagano Prefecture for the first time. E07 has previously been reported in the Tohoku region and Toyama Prefecture. While the STRUCTURE analysis did not reveal any genetic clusters in nuclear DNA, significant spatial genetic structures were identified in both mtDNA and nuclear DNA variations. In addition, we found no evidence of a recent bottleneck. This study is the first to examine the local genetic structure of the black bears in Nagano Prefecture. Although no genetic clusters were detected in nuclear DNA, the estimated effective population size was not particularly large, underscoring the importance of continued genetic monitoring. These new findings obtained in this study can contribute to anticipating potential conflict zones and guide management and conservation measures in the Kiso region.