A rapid multiplex PCR (m-PCR) method that allows the simultaneous detection, in a single tube, of six commonly encountered waterborne pathogens is developed. The target genes used were: the aerolysin (aero) gene of Aeromonas hydrophila, the invasion plasmid antigen H (ipaH) gene of Shigella flexneri, the attachment invasion locus (ail) gene of Yersinia enterocolitca, the invasion plasmid antigen B (ipaB) gene of Salmonella typhimurium, the enterotoxin extracellular secretion protein (epsM) gene of Vibrio cholerae and a species-specific region of the 16S-23S rDNA (Vpara) gene of Vibrio parahaemolyticus were used as the gene targets. Multiplex PCR using the six pairs of primers produced specific amplicons of the expected sizes from mixed populations of reference bacterial strains in seawater and from pure cultures. The m-PCR assay was specific and rapid, with a turnaround time of < 12 h. The detection limit of the assay for the bacterial targets was estimated at 10(0)-10(2) cfu. Multiplex PCR analysis was performed on 19 seawater samples collected around Hong Kong and the results indicated significant levels of four bacterial pathogens at several sites where primary sewage wastes are discharged, and the levels of which showed no correlation with E. coli counts. Overall, both laboratory and field validation results demonstrated that the m-PCR assay developed in this study could provide a cost-effective and informative supplement to conventional microbiological methods for routine monitoring and risk assessment of water quality.