We assessed the North American mid-continent great rivers (Upper Mississippi, Missouri, and Ohio). We estimated the extent of each river in most- (MDC) or least-disturbed condition (LDC) based on multiple biological response indicators: fish and macroinvertebrate, trophic state based on chlorophyll a, macrophyte cover, and exposure of fish-eating wildlife to toxic contaminants in fish tissue (Hg, total chlordane, total DDT, PCBs). We estimated the extent of stressors on each river including nutrients, suspended solids, sediment toxicity, invasive species, and land use (agriculture and impervious surface). All three rivers had a greater percent of their river length in MDC than in LDC based on fish assemblages. The Upper Mississippi River had the greatest percent of river length with eutrophic status. The Ohio River had the greatest percent of river length with fish with tissue contaminant levels toxic to wildlife. Overall, condition indices based on fish assemblages were more sensitive to stress than macroinvertebrate indices. Compared to the streams in its basin, more of the Upper Mississippi and Missouri Rivers were in MDC for nutrients than the Ohio River. Invasive species (Asian carp and Dreissenid mussels) were less widespread and less abundant on the Missouri River than on the other great rivers. The Ohio River had the most urbanized floodplains (greatest percent impervious surface). The Missouri River had the most floodplain agriculture. The effect of large urban areas on river condition was apparent for several indicators. Ecosystem condition based in fish assemblages, trophic state, and fish tissue contamination was related to land use on the floodplain and at the subcatchment scale. This is the first unbiased bioassessment of the mid-continent great rivers in the United States. The indicators, condition thresholds, results, and recommendations from this program are a starting point for improved future great river assessments.