Diarrheagenic Escherichia coli strains have emerged as foodborne pathogens of worldwide public health importance. In this study, a one-shot multiplex PCR (mPCR) was developed for simultaneous detection of four virulence genes each specific for a strain of diarrheagenic E. coli. The mPCR products were separated and detected by DHPLC. The four diarrheagenic E.coli strains all showed unique peak patterns as their "molecular fingerprints" in the chromatogram. With the reference strains, mPCR-DHPLC showed a high sensitivity detecting as few as 8, 6, 7 and 6 colony-forming units (CFU) for enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and enteroinvasive E.coli (EIEC), respectively, in each miniliter of bacteria cultured in the enrichment broth for 18 h. Our study demonstrated the reproducibility, sensitivity and reliability of the mPCR-DHPLC system in the simultaneous amplifications of different virulence genes to differentiate different diarrheagenic E. coli strains, as well as the feasibility of adaptation to a high throughput platform for the large scale screening of causal pathogens present in food and other industrial products.