Within the Athabasca River basin, five genetically differentiated clusters were found. The Saskatchewan and Athabasca River basins contained similar levels of heterozygosity but were differentiated from one another. Across this region, we sampled 431 Bull Trout from 20 sites in the Athabasca and Saskatchewan River basins and assayed 10 microsatellite loci to characterize within‐ and among‐population genetic variation. In Alberta's Eastern Slope region, populations at the range margin have progressively been lost, motivating us to better understand the amount and distribution of genetic variation in headwater habitats and some downstream sites where they continue to persist. Across its native range, Bull Trout ( Salvelinus confluentus) extent and abundance are in decline due to historic overharvest, invasive non‐native species, and habitat loss. Because reductions in population size and extent can negatively impact genetic diversity and gene flow, which are vital for sustained local adaptation, it is important to measure these characteristics in threatened species that may yet be rescued from extinction. Freshwater ecosystems are negatively impacted by a variety of anthropogenic stressors, with concomitant elevated rates of population decline for freshwater aquatic vertebrates.