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Contaminated agricultural water has been a source of pathogenic Escherichia coli in recent produce-related outbreaks. The purpose of this study was to characterize E. coli isolates from agricultural water sources by using whole-genome sequencing (WGS) to better understand contamination routes. Groundwater and surface water samples were collected quarterly from five farms in Missouri and Kansas over a 1-year period. Samples were tested for generic E. coli by using U.S. Environmental Protection Agency Method 1603, and presumptive E. coli colonies were isolated. In total, 570 isolates were analyzed by PCR, with 191 of these isolates confirmed as E. coli. WGS was completed using an Illumina MiSeq system. The de novo genome assemblies were obtained with Shovill pipeline version 0.9. The NCBI Pathogen Detection system was used to identify antimicrobial resistance (AMR) genes. The prevalence of E. coli was higher during spring and summer than winter. A diverse serotype pool was observed where more than 53% of isolates could be linked to a bovine source as the potential animal host. An AMR analysis showed that 100% of isolates carried at least two antimicrobial resistance genes. Recognizing the diversity of E. coli may help guide agricultural water assessments as proposed in the new agricultural water rule Food Safety Modernization Act Produce Safety Rule. [ABSTRACT FROM AUTHOR]