Fish assemblages and environmental variables were evaluated from 37 least-disturbed, 1st- through 6th-order streams and springs in the upper Snake River basin, western USA. Data were collected as part of the efforts by the U.S. Geological Survey National Water Quality Assessment Program and the Idaho State University Stream Ecology Center to characterize aquatic biota and associated habitats in least-disturbed coldwater streams. Geographically, the basin comprises four ecoregions. Environmental variables constituting various spatial scales, from watershed characteristics to instream habitat measures, were used to examine distribution patterns in fish assemblages. Nineteen fish species in the families Salmonidae, Cottidae, Cyprinidae, and Catostomidae were collected. Multivariate analyses showed high overlap in stream fish assemblages among the ecoregions. Major environmental factors determining species distributions in the basin were stream gradient, watershed size, conductivity, and percentage of the watershed covered by forest. Lowland streams (below 1,600 m in elevation), located mostly in the Snake River Basin/High Desert ecoregion, displayed different fish assemblages than upland streams (above 2,000 m elevation) in the Northern Rockies, Middle Rockies, and Northern Basin and Range ecoregions. For example, cottids were not found in streams above 2,000 m in elevation. In addition, distinct fish assemblages were found in tributaries upstream and downstream from the large waterfall, Shoshone Falls, on the Snake River. Fish metrics explaining most of the variation among sites included the total number of species, number of native species, number of salmonid species, percent introduced species, percent cottids, and percent salmonids. Springs also exhibited different habitat conditions and fish assemblages than streams. The data suggest that the evolutionary consequences of geographic features and fish species introductions transcend the importance of ecoregion boundaries on fish distributions in the upper Snake River basin.