Abstract: Objective To establish a rapid and specific multiplex polymerase chain reaction assay for the detection of diffuse adherent Escherichia coli (DAEC) and understand the prevalence of DAEC infection in diarrhea patients in China. Methods The multiplex PCR assay was established based on four adhesion-associated genes, i.e. afaB, afaC, afaD, daaE and 16S rRNA gene was used as control. The assay was evaluated with other E. coli strains and enteric pathogens. A total of 389 stool samples from diarrhea patients were screened by the multiplex PCR assay. Results The multiplex PCR assay could amplify the related genes and the sensitivity was 3.20 101 CFU/reaction or 8.01103 CFU/ml for pure culture. The detection rate of DAEC in diarrhea patients was 6.2%. Conclusion The established multiplex PCR assay is specific suitable for screening and detection of DAEC.