The Asian black bear inhabiting the Shimokita Peninsula, the northernmost part of Honshu, Japan, is categorized as one of the endangered local populations (LPs) in the list of threatened species under the Endangered Species Act. The Shimokita population has become geographically isolated and fragmented due to recent human activity. In this study, we conducted DNA sampling between 2005–2007 of two neighboring bear populations across two regions, Shimokita and Tsugaru, and analyzed the genetic characteristics from the mitochondrial control region (approximately 700 bp) and seven microsatellite loci. Based on the analysis of both microsatellite markers and the mitochondrial control region, we found that the genetic diversity of the Shimokita population was lower than that of the Tsugaru population. An assignment test showed that three of 112 individual bears migrated between subpopulations, suggesting the low level of gene flow between subpopulations. The extent of genetic differentiation between populations (Fst) was 0.163 in microsatellites and 0.443 in mtDNA. These findings indicate that the Shimokita population is genetically isolated from other populations by habitat fragmentation due to human activity and historical events.