Brook trout (Salvelinus fontinalis) is the only salmonid native to the southern Appalachian Mountains. The range of brook trout within this region was greatly reduced during the 20th century due to environmental degradation and the introduction of non-native rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta). Efforts to supplement trout populations and to repopulate streams in which trout had been extirpated also included stocking of hatchery-reared brook trout, the stocks for which originated from northern populations. Recently, molecular genetic analyses have demonstrated there to be distinct differences between brook trout native to the southern Appalachians and those found north of this region. In the present study, the genetic origin was determined for wild brook trout populations within 37 streams in the upper French Broad River system, Transylvania and Henderson counties, North Carolina. Fish were collected by electroshocking and muscle tissue was obtained non-lethally from each. The tissues were analyzed by cellulose acetate gel protein electrophoresis for creatine kinase and 5 other potentially informative enzymes. In only 6 streams (16%) were the brook trout of the unaltered native Southern Appalachian strain. Seven streams (19%) contained brook trout of solely northern hatchery-derived origin, and 24 streams (65%) contained brook trout of mixed genetic origin. Results indicate that stocking of hatchery brook trout into streams within the French Broad River system has led to widespread establishment of non-native northern strain brook trout or interbreeding between northern strain and Southern Appalachian strain brook trout to produce populations of mixed genetic origin.