Water samples from the Platte River were collected and analyzed for environmental DNA (eDNA). The U.S. Geological Survey (USGS) Nebraska Water Science Center (NEWSC) collected samples monthly from November 2019 to September 2022 at the Platte River near Ashland, Nebraska (USGS: 06801000). The USGS, in cooperation with the City of Lincoln, collected these samples. Water from the Platte River was collected as a series of weighted bottle subsamples that were taken from equal width increments across the river and composited into a sterilized 1-liter bottle. An aliquot of this composited sample was extracted using a sterile syringe and then filtered by the USGS NEWSC staff using filters provided by Jonah Ventures. These filters were frozen and shipped to Jonah Ventures Laboratory in Boulder, Colorado. Any eDNA associated with 6 vertebrates (bovine, human, poultry, swine, canine, or sheep) or the Escherichia Coli bacteria were then extracted from the filter and processed using quantitative polymerase chain reaction (qPCR). These qPCR assays were chosen to identify and quantify the relative amount of eDNA for these different species that are present in the river near Ashland, Nebraska. Midway through the study effort (August 2021), a new preservative was included in the analytical method which may have introduced a step trend in some of the qPCR results. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.