Using mitochondrial DNA (mtDNA) restriction analysis and starch‐gel electrophoresis of the CK‐A2 locus product, we examined genetic variation in 311 brook trout (Salvelinus fontinalis) from 11 native, 5 hatchery‐derived, and 8 hybrid populations in the southern Appalachian Mountains. Native southern Appalachian fish were genetically distinct from hatchery‐derived fish. Southern and hatchery‐derived fish were reliably distinguished based on three mtDNA restriction sites. Native southern haplotypes differed from hatchery‐derived haplotypes by an average of 0.84%. Northern hatchery‐derived haplotypes varied little in mtDNA compared to native southern haplotypes. Introgression of mtDNA haplotypes and the CK‐A2 locus varied among populations, and introgression of allozyme and mtDNA markers was positively correlated. Continued introductions of nonnative strains of brook trout in the southern Appalachians could simplify the genetic structure of native brook trout populations and eliminate unique genotypes.

Previous research has indicated that native southern Appalachian brook trout Salvelinus fotinalis are genetically distinct from hatchery stocks derived from northeastern populations. Six diagnostic allozyme loci identified in earlier research were used to assess the genetic origin of 38 Tennessee brook trout populations outside of Great Smoky Mountains National Park. Twenty‐two of these populations (58%) were putatively native, eight (21%) were derived from hatchery stocks, and eight (21 %) were hybrids. Significant genetic differences among the 22 native populations were observed, and genetic structure among these populations was high (genetic variance index FST = 0.622). Thirty‐two percent of the genetic variation among native populations was attributable to differences within watersheds, whereas 29% was attributable to variation among the five major watersheds containing brook trout. Populations located north (19) and south (3) of the French Broad River clustered separately, based on a genetic distance index. Knowledge of the genetic characteristics of brook trout populations will enable fisheries managers to make more informed decisions about this resource in Tennessee and elsewhere in the southern Appalachians. Given that maintaining the genetic integrity of native southern Appalachian brook trout is an important goal, our findings will help managers to design strategies that require stocking or stock transfers to create or expand populations.