Steelhead, Oncorhynchus mykiss, were first introduced into the Great Lakes in the late 1800s. Subsequently, natural recruitment across the Lake Michigan basin has been regularly supplemented by primarily one hatchery strain. Recently, multiple strains derived from locations across the species native range along the west coast of the United States have also been stocked by different management agencies. Prior to 1983, hatchery supplementation of Lake Michigan steelhead populations in Michigan was largely unsuccessful due to low smolting rates of small (< 120 mm) hatchery yearlings (estimated survival 0.01%). Accordingly, contributions of hatchery fish to historical adult spawning runs in Michigan tributaries were low (0–30%) across six major drainages. Large yearlings of different hatchery strains (> 150 mm) have been stocked exclusively since 1983, increasing estimates of survival to smolting (90%). Consequently, the proportion of hatchery adults in spawning runs increased to 13–79%. We examined the effects of changes in stocking practices on straying rates of hatchery steelhead and to temporal changes in levels of genetic diversity and relationships among populations. We used microsatellite loci to estimate allele frequencies for six populations sampled for two time periods (1983–1984 and 1998–1999). Measures of inter-population divergence (mean FST) were not significant for either time period. However, spatial genetic relationships among historical and contemporary populations were significantly correlated with geographic distance; a result not expected if gene flow (natural straying) among populations was mediated solely by hatchery supplementation. Increased numbers of alleles in spawning adults from populations can be attributed to alleles specific to recently introduced hatchery strains.