The fluvial Arctic grayling Thymallus arcticus is restricted to less than 5% of its native range in the contiguous United States and was relisted as a category 3 candidate species under the U.S. Endangered Species Act in 2010. Although fluvial Arctic grayling of the lower Gibbon River, Yellowstone National Park, Wyoming, were considered to have been extirpated by 1935, anglers and biologists have continued to report catching low numbers of Arctic grayling in the river. Our goal was to determine whether a viable population of fluvial Arctic grayling persisted in the Gibbon River or whether the fish caught in the river were downstream emigrants from lacustrine populations in headwater lakes. We addressed this goal by determining relative abundances, sources, and evidence for successful spawning of Arctic grayling in the Gibbon River. During 2005 and 2006, Arctic grayling comprised between 0% and 3% of the salmonid catch in riverwide electrofishing (mean < 1%; SE < 1%) and snorkeling (mean = 1%; SE = 1%) surveys; Arctic grayling constituted 0–14% of the salmonid catch obtained by targeted angling (3 of 22 fish; mean = 4%; SE = 5%). Low values of the genetic differentiation index (F ST = 0.0021 ± 0.002 [mean ± 95% confidence interval]) between headwater lake and river Arctic grayling indicated that fish from throughout the Gibbon River system probably belonged to the same population. Back-calculated lengths at most ages were similar among all fish, and successful spawning within the Gibbon River below the headwater lakes was not documented. Few Arctic grayling adults and no fry were detected in the Gibbon River, implying that a reproducing fluvial population does not exist there. These findings have implications for future Endangered Species Act considerations and management of fluvial Arctic grayling within and outside of Yellowstone National Park. Our comprehensive approach is broadly applicable to the management of sparsely detected aquatic species worldwide.