Objective To establish a PCR- DHPLC (polymerase chain reaction-denaturing high-performance liquid chromatography) method for the detection of five foodborne pathogenic bacteria (Salmonella spp., Vibrio parahaemolyticus, Escherichia coli O157:H7, and Listeria monocytogenes). Method The primer sets for the conserved region of 16S rRNA gene were designed and used for PCR amplification. PCR products were detected by DHPLC, and the sensitivity and specificity of this method were tested as well. Results The five PCR products were shown as specific peak profiles with the retention time of 7 min at an oven temperature of 61.4°C. The detection limits of Salmonella spp., Vibrio parahaemolyticus, and Listeria monocytogenes were 5-10 CFU/ml, while that of Shigella flexneri and Escherichia coli O157:H7 were 1-5 CFU/ml. All 83 target bacteria isolates tested were correctly identified and all 38 non-target strains tested were negative. The five pathogens in artificially contaminated food samples were also correctly identified by this method. Conclusion The PCR-DHPLC method was specific and sensitive for the detection of these five bacterial pathogens and could be used for quickly detecting a large number of samples.