Cyanotoxins occur in rivers worldwide but are understudied in lotic ecosystems relative to lakes and reservoirs. We sampled 11 large river sites located throughout the United States during June–September 2017 to determine the occurrence of cyanobacteria with known cyanotoxin-producing strains, cyanotoxin synthetase genes, and cyanotoxins. Chlorophyll a concentrations ranged from oligotrophic to eutrophic (0.5–64.4 µg L⁻¹). Cyanobacteria were present in the algal communities of all rivers (82% of samples, n = 50) but rarely dominated the phytoplankton (0–52% of total abundance; mean = 8.8%). Pseudanabaena and Planktothrix occurred most often, and many (64%) of the cyanobacterial genera identified (n = 25) have known cyanotoxin-producing strains. Cyanotoxin synthetase genes occurred in all but one river. The mcyE and sxtA genes were most common, present in 73% of rivers and 44% and 40% of samples, respectively. The cyrA gene was less common (22% of samples) but occurred in 64% of rivers. The anaC gene was detected in one river (4% of samples). Anatoxin-a and microcystins were detected at low levels (0.10–0.38 µg L⁻¹) in 2 midcontinent rivers. Cylindrospermopsins and saxitoxins were not detected. Cyanobacteria, cyanotoxin synthetase genes, and cyanotoxins were present at low concentrations throughout this subset of US rivers. Eutrophic rivers located in the midcontinent region of the United States had the highest algal biomass, abundance of cyanotoxin synthetase genes, and cyanotoxin occurrence.