A habitat-based aquatic macroinvertebrate study was initiated in the Lower Missouri River to evaluate relative quality and biological condition of dike pool habitats. Water-quality and sediment-quality parameters and macroinvertebrate assemblage structure were measured from depositional substrates at 18 sites. Sediment porewater was analysed for ammonia, sulphide, pH and oxidation–reduction potential. Whole sediments were analysed for particle-size distribution, organic carbon and contaminants. Field water-quality parameters were measured at subsurface and at the sediment–water interface. Pool area adjacent and downstream from each dike was estimated from aerial photography. Macroinvertebrate biotic condition scores were determined by integrating the following indicator response metrics: % of Ephemeroptera (mayflies), % of Oligochaeta worms, Shannon Diversity Index and total taxa richness. Regression models were developed for predicting macroinvertebrate scores based on individual water-quality and sediment-quality variables and a water/sediment-quality score that integrated all variables. Macroinvertebrate scores generated significant determination coefficients with dike pool area (R2 = 0.56), oxidation–reduction potential (R2 = 0.81) and water/sediment-quality score (R2 = 0.71). Dissolved oxygen saturation, oxidation–reduction potential and total ammonia in sediment porewater were most important in explaining variation in macroinvertebrate scores. The best two-variable regression models included dike pool size + the water/sediment-quality score (R2 = 0.84) and dike pool size + oxidation–reduction potential (R2 = 0.93). Results indicate that dike pool size and chemistry of sediments and overlying water can be used to evaluate dike pool quality and identify environmental conditions necessary for optimizing diversity and productivity of important aquatic macroinvertebrates. A combination of these variables could be utilized for measuring the success of habitat enhancement activities currently being implemented in this system. Published in 2011 by John Wiley & Sons, Ltd.