Detecting the environmental DNA (eDNA) of an organism can in principle be easier and more efficient than detecting the organism itself, particularly for rare or cryptic species (Hinlo et al., 2018; Jerde et al., 2011; Pfleger et al., 2016). eDNA detection is particularly valuable for threatened fishes, which are otherwise most often surveyed with labor-intensive capture surveys (e.g., electrofishing, netting), and might be harmed during the capture process (Dolan and

Brook Trout Salvelinus fontinalis have faced significant declines throughout their native range and have been stocked in Midwestern waters since the late 1800s to offset such losses. Several studies have investigated the genetic effects of these stockings, but these efforts have been confined to relatively small spatial scales. In this study, we compiled 8,454 Brook Trout microsatellite genotypes from 188 wild Midwestern populations and 26 hatchery strains to provide novel insights of broadscale population structure, regional patterns of genetic diversity, and estimates of hatchery introgression for inland Wisconsin populations. Our results indicate high levels of differentiation among our study populations, a lack of hydrological population structuring, lower estimates of genetic diversity in the Driftless Area, and that hatchery introgression has been largely confined to regions of inland Wisconsin that have been heavily affected by anthropogenic disturbances (i.e., the Driftless Area). We also provide evidence that populations may be able to purge hatchery‐derived alleles, discuss possible mechanisms behind this phenomenon, and consider their relevance to accurate estimation of hatchery introgression. Collectively, these results summarize the genetic effects of over a century of anthropogenic disturbance on native Brook Trout populations and emphasize the importance of integrating historical data into contemporary genetic research of intensively managed species.