Translocation of isolated species into suitable habitats may help secure vulnerable, geographically limited species. Due to the decline of Wyoming Hornyhead Chub Nocomis biguttatus, conservation actions such as translocation of populations within the plausible historical range are being considered to improve population redundancy and resiliency to disturbance events. Translocation of Wyoming Hornyhead Chub must be rigorously evaluated because a hatchery stock does not exist, so all fish used in translocations will come from the wild population. We present an approach to identify best available translocation sites prior to translocation efforts taking place. We evaluated fish community composition and habitat conditions at 54 potential translocation sites for Hornyhead Chub within 12 streams of the North Platte River Basin of Wyoming. We used two analyses to identify translocation sites most similar to currently occupied Hornyhead Chub sites on the Laramie River: hurdle models to predict hypothetical abundance of Hornyhead Chub at translocation sites and non-metric multidimensional scaling (NMDS) with fish community and habitat conditions. Presence and abundance of Hornyhead Chub was related to lack of nonnative predators and habitat features characteristic of backwater and velocity refuge habitats. We used a rank scoring system to weight the outcomes of each analysis and the highest ranking translocation sites occurred at a historical locality, the Sweetwater River. Our approach may be appropriate for other at-risk species with isolated distributions and little historical data.