We evaluated the potential of vegetation and sediment habitats in wetlands of the St. Lawrence River for developing a macroinvertebrate bioassessment program with reference conditions. During September 2004, we collected macroinvertebrates in emergent vegetation and sediment in both fluvial sites (reference) and tributary-plume sites (impacted) in waters of the north and south shores of Lake Saint-Pierre (St. Lawrence River). In each habitat, we compared taxa richness, abundance, and community structure of macroinvertebrates between reference and impacted sites, and used multivariate models to relate macroinvertebrate community to environmental conditions. Each habitat was suitable for discriminating reference fluvial sites from impacted tributary-plume sites based on macroinvertebrate communities. In emergent vegetation, macroinvertebrates were dominated by epibenthic fauna such as crustaceans (Gammaridae, Asellidae) and molluscs (Valvatidae) at fluvial sites, and insect larvae (Chironomidae, Caenidae) at tributary-plume sites. In sediment, macroinvertebrates comprised a greater proportion of endobenthic fauna such as Oligochaeta and Sphaeridae. Crustaceans and molluscs were still dominant at fluvial sites and Oligocheata and Chironomidae at tributary-plume sites. No strong difference was depicted in macroinvertebrate composition between north and south shore water masses. Environmental variables explained a higher proportion of variance in macroinvertebrate community composition in emergent vegetation than in sediment (68% versus 44%). Macroinvertebrate composition in sediment was more related to metal contamination, whereas macroinvertebrate composition in emergent vegetation was related primarily to vegetation type and water quality. Relevance of the study for bioassessment of macroinvertebrates in the St. Lawrence River using the reference condition approach is discussed.