Human-driven habitat fragmentation is increasing worldwide, and consequently many wild populations are subdivided, isolated and reduced in size. These changes in population structure reduce dispersal among subpopulations, limiting gene flow, accelerating genetic differentiation, and reducing genetic diversity and effective population sizes. Habitat fragmentation is associated with a reduced ability for populations and species to respond to changing environments, exacerbating extinction risks. The Atlantic-Gaspésie population of woodland caribou (Rangifer tarandus caribou) is isolated and genetically differentiated from other populations in Canada. It has been declining dramatically during the last century and is now considered Endangered. From a management perspective, this population is considered as a single unit of ~ 80 individuals, but GPS telemetry suggests that three subgroups use separate geographical areas and show limited dispersal. In this study, we used 16 microsatellite loci to (1) quantify and compare the genetic diversity observed within the three subgroups of the Atlantic-Gaspésie population, (2) evaluate the extent of the spatiotemporal genetic substructure among them by assessing whether the fine-scale genetic structure differs between subgroups and if it has changed over 15 years, and (3) estimate their effective population size. We found no change in genetic diversity among/within subgroups over time. We detected genetic substructure among subgroups based on their geographical locations (Logan-Albert vs. McGerrigle) and found evidence that this substructure has increased in recent years. The effective population size of this population appears to have declined by 53% over the last 15 years and is now estimated at Ne = 16 individuals. Management plans and conservation actions should consider this spatial genetic substructure to prevent further decline of this endangered population.