The number of sites sampled must be considered when determining the effort necessary for adequately assessing taxa richness in an ecosystem for bioassessment purposes; however, there have been few studies concerning the number of sites necessary for bioassessment of large rivers. We evaluated the effect of sample size (i.e., number of sites) necessary to collect vertebrate (fish and aquatic amphibians), macroinvertebrate, and diatom taxa from seven large rivers in Oregon and Washington, USA during the summers of 2006–2008. We used Monte Carlo simulation to determine the number of sites needed to collect 90–95% of the taxa 75–95% of the time from 20 randomly located sites on each river. The river wetted widths varied from 27.8 to 126.0 m, mean substrate size varied from 1 to 10 cm, and mainstem distances sampled varied from 87 to 254 km. We sampled vertebrates at each site (i.e., 50 times the mean wetted channel width) by nearshore-raft electrofishing. We sampled benthic macroinvertebrates nearshore through the use of a 500-μm mesh kick net at 11 systematic stations. From each site composite sample, we identified a target of 500 macroinvertebrate individuals to the lowest possible taxon, usually genus. We sampled benthic diatoms nearshore at the same 11 stations from a 12-cm2 area. At each station, we sucked diatoms from soft substrate into a 60-ml syringe or brushed them off a rock and rinsed them with river water into the same jar. We counted a minimum of 600 valves at 1,000× magnification for each site. We collected 120–211 diatom taxa, 98–128 macroinvertebrate taxa, and 14–33 vertebrate species per river. To collect 90-95% of the taxa 75-95% of the time that were collected at 20 sites, it was necessary to sample 11–16 randomly distributed sites for vertebrates, 13–17 sites for macroinvertebrates, and 16–18 sites for diatoms. We conclude that 12–16 randomly distributed sites are needed for cost-efficient sampling of vertebrate richness in the main stems of our study rivers, but 20 sites markedly underestimates the species richness of benthic macroinvertebrates and diatoms in those rivers.