One of the dilemmas in designing any large-scale macroinvertebrate bioassessment is deciding where to sample within streams. Streams contain a wide variety of habitats with varying macroinvertebrate assemblages, yet consistency in sampling protocol is needed to interpret results across sites in a region. The Environmental Monitoring and Assessment Program (EMAP) conducted large regional probability surveys in the mid-Atlantic (1993–1998) and the western US (2000–2001). In these surveys, 2 macroinvertebrate sample types were collected at each site: pool and riffle in the mid-Atlantic region, and reachwide and riffle in the western US. We analyzed data from sites where both types of samples had been collected (206 mid-Atlantic and 293 western US sites) to examine the effects of sample type on typical metric and multivariate analyses done in bioassessments. Sample types differed in terms of taxon richness measures and assemblage composition, and differences were more pronounced between mid-Atlantic riffle and pool samples than between western US reachwide and riffle samples. Nonetheless, sample-type differences did not obscure the overall pattern in ordination analyses, nor did they influence detection of important environmental gradients. In addition, bioassessments based on Ephemeroptera, Plecoptera, and Trichoptera taxon richness showed that regional assessments differed little with sample type. Our analyses indicate that typical bioassessment methods are relatively robust with respect to sample type in regional surveys. Reachwide sampling could be used with little risk to the quality of assessments in surveys covering large geographic areas where a single targeted habitat may not occur at all sites. Reachwide sampling is also easy to apply consistently at most sites and requires approximately the same effort and cost as single-habitat sampling.