We conducted an aquatic macroinvertebrate assessment in the channelized reach of the lower Missouri River, and used statistical analysis of individual metrics and multimetric scores to identify community response patterns and evaluate relative biological condition. We examined longitudinal site differences that are potentially associated with water qualityrelated factors originating from the Kansas City metropolitan area, using data from coarse rock substrate in flowing water habitats (outside river bends), and depositional mud substratein slack water habitats (dike fields). Three sites above rivermile (RM) 369 in Kansas City (Nebraska City, RM = 560; St. Joseph, RM = 530; Parkville, RM = 377) and three below (Lexington, RM = 319; Glasgow, RM = 228; Hermann, RM = 94) were sampled with rock basket artificial substrates, a qualitative kicknet method, and the Petite Ponar. We also compared the performance of the methods used. A total of 132 aquatic macroinvertebrate taxa were collected from the lower Missouri River; one third of these taxa belonged to the sensitiveEPOT insect orders (Ephemeroptera, Plecoptera, Odonata, and Trichoptera). Rock baskets had the highest mean efficiency (34.1%) of the methods, and the largest number of taxa was collected by Ponar (n = 69) and kicknet (n = 69) methods. Seven of the 15 metrics calculated from rock basket data, and five ofthe nine metrics calculated from Ponar data showed highly significant differences (ANOVA, P < 0.001) at one or more sitesbelow Kansas City. We observed a substantial reduction in net-spinning Trichoptera in rock habitats below Kansas City (Lexington), an increase in relative dominance of Oligochaeta in depositional habitats at the next site downstream (Glasgow), and lower relative condition scores in rock habitat at Lexingtonand depositional habitat at Glasgow. Collectively, these data indicate that some urban-related impacts on the aquatic macroinvertebrate community are occurring. Our results suggest that the methods and assessment framework we used in this studycould be successfully applied on a larger scale with concurrentwater and sediment chemistry to validate metrics, establish impairment levels, and develop a specific macroinvertebrate community index for the lower Missouri River. We recommend accomplishing this with longitudinal multi-habitat sampling at a larger number of sites related to all potential sources of impairment, including major tributaries, urban areas, and point sources.