Quantitative polymerase chain reaction (qPCR) data were compiled for the biosynthesis genes of five toxins of interest produced by cyanobacteria as well as an internal control. Sample deoxyribonucleic acid (DNA) were collected from 21 sites which includes 17 sites from Lake Okeechobee, and four sites from the Caloosahatchee River and the St. Lucie River. Samples were collected on a monthly basis from March 2019 through October 2021, provided water levels were high enough for a sample to be collected. Samples from Lake Okeechobee were collected by the South Florida Water Management District (SFWMD) and samples from the Caloosahatchee and St. Lucie Rivers were collected by the U.S. Geological Survey (USGS). Environmental DNA was extracted soon after collection and samples were analyzed via qPCR throughout the sampling period. Each qPCR assay tests for a different cyanotoxin biosynthesis gene with one assay testing for cyanobacterial 16S DNA as an internal control. The five toxin biosynthesis genes tested were microcystin (mcyE), nodularin (ndaF), saxitoxin (sxtA), anatoxin (anaC), and cylindrospermopsin (cyrA). Results are reported in the number of gene copies per microliter and represent the cyanotoxin biosynthesis gene concentrations in the Lake Okeechobee watershed for the study period.