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Chemical and microbiological results, quality assurance and quality control, site location, and method information for surface water, bed sediment, and wastewater effluent collected from 34 stream locations across Iowa (United States). Environmental samples were analyzed for a suite of 29 antibiotics, plated on selective media for 15 types of bacteria growth, and DNA was extracted from culture growth and used in downstream polymerase chain reaction (PCR) assays for the detection of 24 antibiotic resistance genes. Environmental field data were collected in-situ for water temperature (°C), specific conductivity (µs/cm), dissolved oxygen (mg/L), pH (standard units), and turbidity (FNU) using a multiparameter sonde...