We measured mercury (Hg) concentrations in whole fish from the Upper Mississippi, Missouri, and Ohio Rivers to characterize the extent and magnitude of Hg contamination and to identify environmental factors influencing Hg accumulation. Concentrations were generally lower (80% of values between 20?200 ng g¹ wet weight) than those reported for other regions (e.g., upper Midwest and Northeast U.S.). Mercury exceeded the risk threshold for belted kingfisher (Ceryle alcyon, the most sensitive species considered) in 33?75% of river length and 1?7% of river length for humans. Concentrations were lower in the Missouri than in the Mississippi and Ohio Rivers, consistent with continental-scale patterns in atmospheric Hg deposition. Body size and trophic guild were the best predictors of Hg concentrations, which were highest in large-bodied top predators. Site geochemical and landscape properties were weakly related with fish Hg. Moreover, relationships often ran contrary to conventional wisdom, and the slopes of the relationships (positive or negative) were inconsistent among fish guilds and rivers. For example, sulfate is positively associated with fish Hg concentrations but was negatively correlated with Hg in five of six regression models of tissue concentrations. Variables such as pH, acid neutralizing capacity, and total phosphorus did not occur at levels associated with high fish Hg concentrations, partially explaining the relatively low Hg values we observed. ?? 2010 American Chemical Society.