Determining adequate sampling effort for characterizing fish assemblage structure in nonwadeable rivers remains a critical issue in river biomonitoring. Two-pass boat electrofishing data collected from 500-1,000-m-long river reaches as part of the U.S. Geological Survey's National Water-Quality Assessment (NAWQA) Program were analyzed to assess the efficacy of single-pass boat electrofishing. True fish species richness was estimated by use of a two-pass removal model and nonparametric jackknife estimation for 157 sampled reaches across the United States. Compared with estimates made with a relatively unbiased nonparametric estimator, estimates of true species richness based on the removal model may be biased, particularly when true species richness is greater than 10. Based on jackknife estimation, the mean percent of estimated true species richness collected in the first electrofishing pass (p??j,s1) for all 157 reaches was 65.5%. The effectiveness of single-pass boat electrofishing may be greatest when the expected species richness is relatively low (>10 species). The second pass produced additional species (1-13) in 89.2% of sampled reaches. Of these additional species, centrarchids were collected in 50.3% of reaches and cyprinids were collected in 45.9% of reaches. Examination of relations between channel width ratio (reach length divided by wetted channel width) and p??j,s1 values provided no clear recommendation for sampling distances based on channel width ratios. Increasing sampling effort through an extension of the sampled reach distance can increase the percent species richness obtained from single-pass boat electrofishing. When single-pass boat electrofishing is used to characterize fish assemblage structure, determination of the sampling distance should take into account such factors as species richness and patchiness, the presence of species with relatively low probabilities of detection, and human alterations to the channel.