Biologists are often faced with the difficult decision in managing native salmonids of where and when to install barriers as a conservation action to prevent upstream invasion of nonnative fishes. However, fine-scale approaches to assess long-term persistence of populations within streams and watersheds chosen for isolation management are often lacking. We employed a spatially-explicit approach to evaluate stream habitat conditions, relative abundance, and genetic diversity of native westslope cutthroat trout (Oncorhynchus clarkii lewisi) within the Akokala Creek watershed in Glacier National Park- a population threatened by introgressive hybridization with nonnative rainbow trout (O. mykiss) from nearby sources. The systematic survey of 24 stream reaches showed broad overlap in fish population and suitable habitat characteristics among reaches and no natural barriers to fish migration were found. Analysis of population structure using 16 microsatellite loci showed modest amounts of genetic diversity among reaches, and that fish from Long Bow Creek were the only moderately distinct genetic group. We then used this information to assess the potential impacts of three barrier placement scenarios on long-term population persistence and genetic diversity. The two barrier placement scenarios in headwater areas generally failed to meet general persistence criteria for minimum population size (2,500 individuals, Ne = 500), maintenance of long-term genetic diversity (He), and no population subdivision. Conversely, placing a barrier near the stream mouth and selectively passing non-hybridized, migratory spawners entering Akokala Creek met all persistence criteria and may offer the best option to conserve native trout populations and life history diversity. Systematic, fine-scale stream habitat, fish distribution, and genetic assessments in streams chosen for barrier installation are needed in conjunction with broader scale assessments to understand the potential impacts of using barriers for conservation of native salmonid populations threatened by nonnative fish invasions.