The Harney Basin is Oregon's largest internally draining basin and contains a unique native fish assemblage. The first and only comprehensive study of the origin and distribution of Harney Basin fishes occurred in 1971. Here, we update this study and identify potential threats to the basin's native fishes a half century later. Our assessment documents that all species of native fishes are still present, and with some exceptions distributed as originally reported. The distinctive fish assemblages in each of the 3 major subbasins within the Harney Basin (Silvies River, Silver Creek, and Donner und Blitzen River) support the hypothesis that terminal lakes in the Harney Basin (Malheur, Mud, and Harney Lakes) act as dispersal barriers, at least for native species. More than half of the fish species in the basin at present are introduced, primarily inhabiting low-elevation sites. We suggest that native non-salmonid species cohabiting these sites face the most immediate threats from climate change, water demands, and impacts from introduced fishes. Overall, our results show that native fish are still relatively widespread across the Harney Basin, but also face increasing threats despite the basin having experienced less development than many other areas in the Pacific Northwest. Improved understanding of the unique fishes of the Harney Basin, along with more detailed assessments of likely future trajectories of environmental risks are proactive measures that could improve conservation outcomes.