I investigated the population and landscape genetics of Arctic Grayling (Thymallus arcticus) distributed throughout several connected river systems in Alberta, Canada. Broad- and fine-scale population structure was examined by genotyping nine microsatellite loci in 1,116 Arctic Grayling captured from 40 sites in the Hay River, Peace River, and Athabasca River basins. Genetic diversity tended to decline from north to south (allele richness-latitude: Spearman’s rank correlation rs = 0.793, P < 0.05), with the lowest level detected in a stocked population. Significant genetic divergence between and within major river basins was found (overall FST (θ) = 0.13) as well as strong isolation by distance patterns in the Peace River basin (Mantel r = 0.97, P < 0.001) and Athabasca River basin (Mantel r = 0.95, P < 0.001). Evidence for gene flow among sites in neighbouring rivers (i.e., 25–100 km apart) was common; significant genetic differentiation tended to occur at the sub-basin level. Allelic richness (Ar) was associated with variables describing post-glacial colonization route, spatial position in the stream network, and density of anthropogenic disturbance. These findings have important implications for species management and conservation, particularly in regards to management unit delineation, supplementation procedures, conservation priorities (i.e., protecting small and/or isolated stocks), and land-use planning.