We genotyped Arctic grayling ( Thymallus arcticus ) at 10 microsatellite loci in 18 samples (n = 726) from Montana, Wyoming, and Saskatchewan to determine genetic relationships among native, captive, and naturalized populations in the upper Missouri River basin, to assess patterns in genetic diversity, and to infer recent demographic histories. Substantial genetic subdivision was observed among sample populations (global FST = 0.10). Canadian populations have been isolated from Missouri River populations long enough for mutation to cause genetic differences between regions (mean pairwise FST = 0.18, RST = 0.54). Within the Missouri River basin, most naturalized lacustrine populations traced their ancestry to Red Rock lakes. Two populations in headwater lakes within the Big Hole River watershed appear to be native. We found neither evidence for introgression of Canadian-origin grayling nor any effect of hatchery stocking in native populations. The native fluvial Big Hole River group was genetically distinct and most diverse (HE = 0.89), whereas native Madison River and Red Rock lakes populations exhibited lower genetic diversity (HE = 0.74 and 0.80, respectively) and evidence of recent bottlenecks. The existing Big Hole and Red Rock populations are at low abundance but do not appear to be at immediate risk of inbreeding depression (Ne = 207.7–228.2).