Diarrheagenic Escherichia coli (DEC) is a group of the most common agents of diarrhea. Highly virulent DEC strains could cause illness with dozens of organisms. Waterborne DEC may be detected using polymerase chain reaction (PCR); however, environmental contaminants can interfere with PCR reaction, thus causing the prevalence of DEC to be underestimated. In this study, we propose an approach to efficiently quantify trace amounts of DEC. An enrichment procedure was performed to amplify total E. coli including DEC in the water samples. By normalizing the number of pathotype-specific genes to the amplification rate of a housekeeping gene in all E. coli, the quantity of DEC in original samples could be assessed. This method allows detection of trace amounts of DEC in receiving waters. The results showed that the presence of DEC in water samples was partially associated with riverside settlement. The DEC concentration was substantially higher at a few sampling sites, suggesting that evaluation of DEC along the river may help identify previously unknown pollution sources. Although the sustainability of DEC in the receiving waters may be low, the risk of DEC infection from the watershed warrants further examination.