We investigated the population 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, Peace, and Athabasca River basins. Genetic diversity tended to decline from north to south (allelic richness versus latitude: Spearman's rank correlation rs = 0.793, P < 0.05); the lowest level of diversity was detected in a stocked population. We found significant genetic divergence between and within major river basins (overall genetic differentiation index FST [θST] = 0.13) and strong isolation-by-distance patterns in the Peace River basin (Mantel's r = 0.97, P < 0.001) and Athabasca River basin (Mantel's r = 0.95, P < 0.001). Evidence for gene flow among sites in neighboring rivers (i.e., 25–100 km apart) was common; significant genetic differentiation tended to occur at the subbasin level. The spatial scale of differentiation for Arctic Grayling is intermediate to those reported for other sympatric salmonid species that differ in population size and degree of spawning site fidelity.