Although altered streamflow has been implicated as a major factor affecting fish assemblages, understanding the extent of streamflow alteration has required quantifying attributes of the natural flow regime. We used predictive models to quantify deviation from expected natural streamflow variability for streams in the eastern USA. Sites with >25% change in mean daily streamflow variability compared with what would be expected in a minimally disturbed environment were defined as having altered streamflow variability, based on the 10th and 90th percentiles of the distribution of streamflow variability at 1279 hydrological reference sites. We also used predictive models to assess fish assemblage condition and native species loss based on the proportion of expected native fish species that were observed. Of the 97 sites, 49 (50.5%) were classified as altered with reduced streamflow variability, whereas no sites had increased streamflow variability. Reduced streamflow variability was related to a 35% loss in native fish species, on average, and a >50% loss of species with a preference for riffle habitats. Conditional probability analysis indicated that the probability of fish assemblage impairment increased as the severity of altered streamflow variability increased. Reservoir storage capacity and wastewater discharges were important predictors of reduced streamflow variability as revealed by random forest analysis. Management and conservation of streams will require careful consideration of natural streamflow variation and potential factors contributing to altered streamflow within the entire watershed to limit the loss of critical stream habitats and fish species uniquely adapted to live in those habitats. Published in 2011 by John Wiley & Sons, Ltd.