AbstractAquatic invertebrates are important components of any aquatic ecosystem and are frequently monitored to determine the ecological integrity of those systems. Various gear types are used to collect aquatic macroinvertebrates in wadeable streams. We analyzed commonly used active and passive gear types, including kick seines, Hess samplers, drift nets, and Hester–Dendy samplers, across a watershed in the Great Smoky Mountains National Park to determine their efficacy under various habitat conditions in wadeable Southern Appalachian mountain streams. Hester–Dendy samplers were significantly different from other gear types for most univariate and multivariate analyses. Kick and Hess samples were most similar in multivariate comparisons of macroinvertebrate assemblages. Drift samples were similar in most univariate analyses but were distinct from other gear types when analyzed with multivariate techniques. Our results indicate that comprehensive assessments of macroinvertebrate assemblages require multiple gear types, various analytical approaches, and consistent taxonomic resolution. The complexity of the fluvial system in question should be thoroughly assessed prior to formulating a macroinvertebrate biomonitoring protocol to better ensure robust and meaningful results.