Human-induced hybridization between fish populations and species is a major threat to aquatic biodiversity worldwide and is particularly relevant to management of the subspecies of cutthroat trout Oncorhynchus clarkii. The upper Snake River basin in Wyoming contains one of the largest remaining populations of Yellowstone cutthroat trout O. clarkii bouvieri, a subspecies of special concern throughout its range; however, little is known about levels of hybridization between Yellowstone cutthroat trout and exotic rainbow trout Omykiss or about the overall genetic population structure for this river basin. There is concern that the Gros Ventre River is a source of hybridization for the Snake River basin. We sampled across the upper Snake River basin to estimate levels of hybridization and population structure and to describe hybrid zone structure and spatial patterns of hybridization throughout the basin. We used this information to help resolve whether the Gros Ventre River was acting as a potential source of hybridization for the upper Snake River basin. We found that Yellowstone cutthroat trout genotypes dominated the river system, but hybridization was detected at low levels in all populations. The Gros Ventre River contained the highest levels of hybridization (population and individual) and displayed evidence of ongoing hybridization between parental genotypes. Levels of hybridization decreased as a function of distance from the Gros Ventre River, suggesting that this population is acting as a source of rainbow trout genes. These patterns were evident despite the fact that levels of genetic connectivity appeared to be higher than those observed in other cutthroat trout populations (global genetic differentiation index FST = 0.04), and we did not find evidence for genetic isolation by distance. Management actions aimed at reducing the presence of highly hybridized cutthroat trout or rainbow trout individuals in the Gros Ventre River will help to maintain the upper Snake River basin as an important conservation area.