Fish mercury concentrations have received considerable attention due to human health implications. Fish mercury concentrations are variable within and among systems due to a suite of biotic and abiotic influences that vary among regions and are difficult to predict. Understanding factors associated with variability in fish mercury concentrations would help guide consumption advisories. Mercury concentrations in channel catfish (Ictalurus punctatus, n = 205), flathead catfish (Pylodictis olivaris, n = 123), northern pike (Esox lucius, n = 60), smallmouth bass (Micropterus dolomieu, n = 176), and walleye (Sander vitreus, n = 176) were assessed in ten Iowa rivers and relationships with land use, water chemistry, and fish characteristics were explored. Mercury concentrations were generally low (mean among all species = 0.17 mg/kg, n = 740) but higher in flathead catfish, northern pike, smallmouth bass, and walleye than channel catfish and were positively related to fish length, age, trophic position, and δ13C signatures. Phosphorus, sulfate, and percent open water and grassland were negatively related to fish mercury concentrations, whereas water hardness, nitrogen-ammonia, Human Threat Index, and percent wetland and forest were positively related to fish mercury concentrations. Fish collected from the Paleozoic Plateau ecoregion in northeast Iowa had higher mercury concentrations than other ecoregions in Iowa. Combined, these factors explained 70% of the variation in fish mercury concentrations. This study provides a comprehensive analysis of abiotic and biotic factors influencing fish mercury concentrations in lotic ecosystems at the individual and system scale that will help guide fish consumption advisories.