As North Carolina’s only native salmonid, Brook Trout Salvelinus fontinalis is a fish of considerable ecological and cultural significance in the state, but anthropogenic alterations to the landscape and introductions of nonnative salmonids have fragmented and reduced its native range. As a result, the North Carolina Wildlife Resources Commission (NCWRC) has enacted numerous efforts to help conserve the species. Annual demographic surveys of self-sustaining Brook Trout populations have been on-going since 1978, which have also included successful efforts to document previously unidentified populations. Beginning in earnest during the 1990s, allozyme testing was used to assess patterns of hatchery introgression, with over 480 collections genotyped at the creatine kinase locus. In 2010, the NCWRC began using microsatellite markers to conduct an extensive survey of Brook Trout genetic diversity and variation. To date, 541 Brook Trout collections representing 11,090 individuals have been genotyped at 12 microsatellite loci. These data have provided insights into evolutionary relationships among populations, spatial patterns of genetic diversity, and the extent of hatchery introgression within populations. Ultimately, increased understanding of genetic diversity and relatedness have been informative for determining that Brook Trout management in North Carolina is likely best enacted at the level of individual populations. Moreover, we have used these data to actively guide stream restoration and population reintroduction activities. Over the last 15 years, NCWRC and its partners have used genetic data to prioritize habitat enhancement activities and guide 17 Brook Trout population reintroduction projects. In the future, we plan to continue expanding the microsatellite genetic baseline while also exploring the utility of phylogenomic analyses to inform Brook Trout conservation activities. Genetic and genomic approaches have great potential to improve the efficacy of conservation actions for Brook Trout in North Carolina and throughout its native range.