Preserving remaining nonhybridized populations Cutthroat Trout Oncorhynchus clarkii is a conservation priority often requiring management action. Although proactive programs for Rainbow Trout O. mykiss and hybrid suppression offer a flexible tool, particularly in large interconnected river basins, this management approach is used less frequently than alternatives such as barriers and piscicides. We describe the results of a targeted Rainbow Trout hybrid suppression program spanning 15 years in the upper Snake River, Wyoming, a core stronghold for Yellowstone Cutthroat Trout O. clarkii bouvieri. Initially, Rainbow Trout hybrids were relatively common in the Gros Ventre River, a major tributary to the Snake River. Between 2002 and 2016, 926 individuals of Rainbow Trout ancestry were removed from the Gros Ventre River. Relative abundance of Rainbow Trout hybrids decreased over this time, while the Yellowstone Cutthroat Trout population increased. Temporal genetic data collected in 2007–2008 and again in 2014 demonstrate that the overall proportion Rainbow Trout admixture and the proportion of hybrids in a sample both significantly decreased in the Gros Ventre River and did not increase elsewhere in the Snake River basin. In conclusion, proactive Rainbow Trout suppression appears to have reduced the threat of Rainbow Trout hybridization in this river basin and helped protect an interconnected metapopulation that has a highly diverse life history and genetic variation important for long-term persistence.