Throughout their range, Brook Trout (Salvelinus fontinalis) occupy thousands of disjunct drainages with varying levels of disturbance, which presents substantial challenges for conservation. Within the southern Appalachian Mountains, fragmentation and genetic drift have been identified as key threats to the genetic diversity of the Brook Trout populations. In addition, extensive historic stocking of domestic lineages of Brook Trout to augment fisheries may have eroded endemic diversity and impacted locally adapted populations. We used 12 microsatellite loci to describe patterns of genetic diversity within 108 populations of wild Brook Trout from Tennessee and used linear models to explore the impacts of land use, drainage area, and hatchery stockings on metrics of genetic diversity, effective population size, and hatchery introgression. We found levels of within-population diversity varied widely, although many populations showed very limited diversity. The extent of hatchery introgression also varied across the landscape, with some populations showing high affinity to hatchery lineages and others appearing to retain their endemic character. However, we found relatively weak relationships between genetic metrics and landscape characteristics, suggesting that contemporary landscape variables are not strongly related to observed patterns of genetic diversity. We consider this result to reflect both the complex history of these populations and the challenges associated with accurately defining drainages for each population. Our study highlights the importance of genetic data to guide management decisions, as complex processes interact to shape the genetic structure of populations and make it difficult to infer the status of unsampled populations.