Characterizing the impacts of hydrologic alterations, pollutants, and habitat degradation on macroinvertebrate species assemblages is of critical value for managers wishing to categorize stream ecosystem condition. A combination of approaches including trait-based metrics and traditional bioassessments provides greater information, particularly in anthropogenic stream ecosystems where traditional approaches can be confounded by variously interacting land use impacts. Macroinvertebrates were collected from two rural and three urban nested study sites in central Missouri, USA during the spring and fall seasons of 2011. Land use responses of conventional taxonomic and trait-based metrics were compared to streamflow indices, physical habitat metrics, and water quality indices. Results show that biotic index was significantly different (p < 0.05) between sites with differences detected in 54 % of trait-based metrics. The most consistent response to urbanization was observed in size metrics, with significantly (p < 0.05) fewer small bodied organisms. Increases in fine streambed sediment, decreased submerged woody rootmats, significantly higher winter Chloride concentrations, and decreased mean suspended sediment particle size in lower urban stream reaches also influenced macroinvertebrate assemblages. Riffle habitats in urban reaches contained 21 % more (p = 0.03) multivoltine organisms, which was positively correlated to the magnitude of peak flows (r² = 0.91, p = 0.012) suggesting that high flow events may serve as a disturbance in those areas. Results support the use of macroinvertebrate assemblages and multiple stressors to characterize urban stream system condition and highlight the need to better understand the complex interactions of trait-based metrics and anthropogenic aquatic ecosystem stressors.