Historic introductions of nonnative rainbow trout Oncorhynchus mykiss into the native habitats of cutthroat trout O. clarkii have impacted cutthroat trout populations through introgressive hybridization, creating challenges and concerns for cutthroat trout conservation. We examined the effects of rainbow trout introductions on the native westslope cutthroat trout O. c. lewisii within the Stehekin River drainage, North Cascades National Park, Washington, by analyzing 1,763 salmonid DNA samples from 18 locations with nine diagnostic nuclear DNA markers and one diagnostic mitochondrial DNA (mtDNA) marker. Pure westslope cutthroat trout populations only occurred above upstream migration barriers in the Stehekin River and Park Creek. Two categories of rainbow trout admixture were observed: (1) less than 10% within the Stehekin River drainage above the Bridge Creek confluence and the middle and upper Bridge Creek drainage and (2) greater than 30% within the Stehekin River below the Bridge Creek confluence and in lower Bridge Creek. Hybrid indices and multilocus genotypes revealed an absence of rainbow trout and reduced hybrid diversity within the Stehekin River above the Bridge Creek confluence relative to hybrid diversity in the Stehekin River below the confluence and within lower Bridge Creek. Cytonuclear disequilibrium statistics revealed assortative mating between westslope cutthroat and rainbow trout but not among hybrids within the same locations. This suggests that a randomly mating hybrid swarm does not currently exist. However, continual migration of parental genotypes into the study location could also create significant cytonuclear disequilibria. The Stehekin River represents a novel and unique example of a dynamic hybridization zone where the invasion of rainbow trout alleles into the Stehekin River westslope cutthroat trout population above the Bridge Creek confluence appears to be impeded, suggesting that divergent ecological or evolutionary mechanisms promote the population structure within the Stehekin River drainage, depending upon location.