Abstract Large river bioassessment protocols lag far behind those of wadeable streams and often rely on fish assemblages of individual rivers. We developed a regional macroinvertebrate index and assessed relative condition of six large river tributaries to the upper Mississippi and Ohio rivers, Midwest USA. In 2004 and 2005, benthic macroinvertebrates, water chemistry, and habitat data were collected from randomly selected sites on each of the St. Croix, Wisconsin, Minnesota, Scioto, Wabash, and Illinois rivers. We first identified the human disturbance gradient using principal components analysis (PCA) of abiotic data. From the PCA, least disturbed sites showed strong separation from stressed sites along a gradient contrasting high water clarity, canopy cover, habitat scores, and plant-based substrates at one end and higher conductivity and nutrient concentrations at the other. Evaluation of 97 benthic metrics identified those with good range, responsiveness, and relative scope of impairment, as well as redundancies with other metrics. The final index was composed of Diptera taxa richness, EPT taxa richness, Coleoptera taxa richness, percent oligochaete and leech taxa, percent collector-filterer individuals, predator taxa richness, percent burrower taxa, tolerant taxa richness, and percent facultative individuals. Each of the selected metrics was scored using upper and lower thresholds based on all sites, and averaging across the nine metric scores, we obtained the Non-wadeable Macroinvertebrate Assemblage Condition Index (NMACI). The NMACI showed a strong response to disturbance using a validation data set and was highly correlated with non-metric multidimensional scaling (NMDS) ordination axes of benthic taxa. The cumulative distribution function of index scores for each river showed qualitative differences in condition among rivers. NMACI scores were highest for the federally protected St. Croix River and lowest for the Illinois River. Other rivers were intermediate and generally reflected the mixture of land use types within individual basins. Use of regional reference sites, though setting a high level of expectation, provides a valuable frame of reference for the potential of large river benthic communities that will aid management and restoration efforts.