The index of biotic integrity (IBI) is a commonly used measure of relative aquatic ecosystem condition; however, its application to coldwater rivers over large geographic areas has been limited. A seven-step process was used to construct and test an IBI applicable to fish assemblages in coldwater rivers throughout the U.S. portion of the Pacific Northwest. First, fish data from the region were compiled from previous studies and candidate metrics were selected. Second, reference conditions were estimated from historical reports and minimally disturbed reference sites in the region. Third, data from the upper Snake River basin were used to test metrics and develop the initial index. Fourth, candidate metrics were evaluated for their redundancy, variability, precision, and ability to reflect a wide range of conditions while distinguishing reference sites from disturbed sites. Fifth, the selected metrics were standardized by being scored continuously from 0 to 1 and then weighted as necessary to produce an IBI ranging from 0 to 100. The resulting index included 10 metrics: number of native coldwater species, number of age-classes of sculpins Cottus spp., percentage of sensitive native individuals, percentage of coldwater individuals, percentage of tolerant individuals, number of alien species, percentage of common carp Cyprinus carpio individuals, number of selected salmonid age-classes, catch per unit effort of coldwater individuals, and percentage of individuals with selected anomalies. Sixth, the IBI responses were tested with additional data sets from throughout the Pacific Northwest. Last, scores from two minimally disturbed reference rivers were evaluated for longitudinal gradients along the river continuum. The IBI responded to environmental disturbances and was spatially and temporally stable at over 150 sites in the Pacific Northwest. The results support its use across a large geographic area to describe the relative biological condition of coolwater and coldwater rivers with low species richness.