Stream habitat restoration and supplemental stocking of hatchery-reared fish have increasingly become key components of recovery plans for imperiled freshwater fish; however, determining when to discontinue stocking efforts, prioritizing restoration areas, and evaluating restoration success present a conservation challenge. In this study, we demonstrate that otolith microchemistry is an effective tool for establishing natal origin of the June Sucker Chasmistes liorus, an imperiled potamodromous fish. This approach allows us to determine whether a fish is of wild or hatchery origin in order to assess whether habitat restoration enhances recruitment and to further identify areas of critical habitat. Our specific objectives were to (1) quantify and characterize chemical variation among three main spawning tributaries; (2) understand the relationship between otolith microchemistry and tributary chemistry; and (3) develop and validate a classification model to identify stream origin using otolith microchemistry data. We quantified molar ratios of Sr:Ca, Ba:Ca, and Mg:Ca for water and otolith chemistry from three main tributaries to Utah Lake, Utah, during the summer of 2013. Water chemistry (log_e transformed Sr:Ca, Ba:Ca, and Mg:Ca ratios) differed significantly across all three spawning tributaries. We determined that Ba:Ca and Sr:Ca ratios were the most important variables driving our classification models, and we observed a strong linear relationship between water and otolith values for Sr:Ca and Ba:Ca but not for Mg:Ca. Classification models derived from otolith element : Ca signatures accurately sorted individuals to their experimental tributary of origin (classification tree: 89% accuracy; random forest model: 91% accuracy) and determined wild versus hatchery origin with 100% accuracy. Overall, this study aids in evaluating the effectiveness of restoration, tracking progress toward recovery, and prioritizing future restoration plans for fishes of conservation concern. Our results have further application, such as identifying subpopulations that provide the greatest reproductive contribution to a metapopulation or finding the reproductive area and origin of invasive fishes.