Abstract 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 F ST = 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 geneti...