Great rivers of the central United States (Upper Mississippi, Missouri, and Ohio rivers) are valuable economic and cultural resources, yet until recently their ecological condition has not been well quantified. In 2004–2005, as part of the Environmental Monitoring and Assessment Program for Great River Ecosystems (EMAP-GRE), we measured legacy organochlorines (OCs) (pesticides and polychlorinated biphenyls, PCBs) and emerging compounds (polybrominated diphenyl ethers, PBDEs) in whole fish to estimate human and wildlife exposure risks from fish consumption. PCBs, PBDEs, chlordane, dieldrin and dichlorodiphenyltrichloroethane (DDT) were detected in most samples across all rivers, and hexachlorobenzene was detected in most Ohio River samples. Concentrations were highest in the Ohio River, followed by the Mississippi and Missouri Rivers, respectively. Dieldrin and PCBs posed the greatest risk to humans. Their concentrations exceeded human screening values for cancer risk in 27–54% and 16–98% of river km, respectively. Chlordane exceeded wildlife risk values for kingfisher in 11–96% of river km. PBDE concentrations were highest in large fish in the Missouri and Ohio Rivers (mean > 1000 ng g−1 lipid), with congener 47 most prevalent. OC and PBDE concentrations were positively related to fish size, lipid content, trophic guild, and proximity to urban areas. Contamination of fishes by OCs is widespread among great rivers, although exposure risks appear to be more localized and limited in scope. As an indicator of ecological condition, fish tissue contamination contributes to the overall assessment of great river ecosystems in the U.S.