Informed conservation of stream fishes requires detailed understanding of the effects of both natural processes and anthropogenic activities on genetic diversity. Brook Trout Salvelinus fontinalis, a salmonid native to eastern North America, typically resides in cold, high-quality stream ecosystems. The species has not only faced historical anthropogenic pressures, but also confronts current and future pressures. In a genetic analysis we used a reduced representation sequencing method (ddRADseq) to characterize 63 individuals from 23 streams where Brook Trout are native in the Appalachian region of Pennsylvania. A total of 2,590 loci passed filtering criteria, and 53% displayed significant association with a major stream drainage basin (Susquehanna or Allegheny; mean FST = 0.085). Mapping of the sequencing reads to the Atlantic Salmon Salmo salar genome revealed no clustering of high interdrainage FST values to specific genome regions. Evidence for genetic heterogeneity within each drainage basin was also detected. Stepwise regression of observed heterozygosity against geographic and environmental features revealed that drainage basin and effective area of watersheds were significant predictors of observed heterozygosity of Brook Trout within streams. Natural features such as waterfalls and major drainage basin, as well as the effects of dams and acid-mine drainage have fragmented habitat and shaped genetic diversity within Brook Trout populations in the Appalachian region of Pennsylvania, overall indicating the vulnerability of this species to increased industrialization.