This U.S. Geological Survey (USGS) data release provides discretely measured cyanotoxin, chlorophyll-a, and cyanobacterial toxin genetic data for samples collected from twelve large river sites throughout the United States, from June through October 2019. Discrete water-quality samples were analyzed for cyanotoxins (anatoxin, cylindrospermopsin, microcystin, and saxitoxin), chlorophyll-a, and cyanobacterial toxin genetics as part of a National Water-Quality Assessment Project pilot study to describe cyanobacteria and cyanotoxin occurrence in the Nation's large rivers. The data release contains the genetic data (in .csv and .xlsx formats), qPCR standard curve information (in .csv and .xlsx formats), and a readme file explaining the data headers for the genetic and standard curve files.