Loss and fragmentation of natural habitats caused by human land uses have subdivided several formerly contiguous large carnivore populations into multiple small and often isolated subpopulations, which can reduce genetic variation and lead to precipitous population declines. Substantial habitat loss and fragmentation from urban development and agriculture expansion relegated the Highlands-Glades subpopulation (HGS) of Florida, USA, black bears (Ursus americanus floridanus) to prolonged isolation; increasing human land development is projected to cause ≥ 50% loss of remaining natural habitats occupied by the HGS in coming decades. We conducted a noninvasive genetic spatial capture-recapture study to quantitatively describe the degree of contemporary habitat fragmentation and investigate the consequences of habitat fragmentation on population density and genetics of the HGS. Remaining natural habitats sustaining the HGS were significantly more fragmented and patchier than those supporting Florida's largest black bear subpopulation. Genetic diversity was low (AR = 3.57; HE = 0.49) and effective population size was small (NE = 25 bears), both of which remained unchanged over a period spanning one bear generation despite evidence of some immigration. Subpopulation density (0.054 bear/km2) was among the lowest reported for black bears, was significantly female-biased, and corresponded to a subpopulation size of 98 bears in available habitat. Conserving remaining natural habitats in the area occupied by the small, genetically depauperate HGS, possibly through conservation easements and government land acquisition, is likely the most important immediate step to ensuring continued persistence of bears in this area. Our study also provides evidence that preferentially placing detectors (e.g., hair traps or cameras) primarily in quality habitat across fragmented landscapes poses a challenge to estimating density-habitat covariate relationships using spatial capture-recapture models. Because habitat fragmentation and loss are likely to increase in severity globally, further investigation of the influence of habitat fragmentation and detector placement on estimation of this relationship is warranted.