Abstract: Objective To establish a rapid multiplex PCR assay to identify and differentiate the five main pathotypes of diarrheogenic Escherichia coli and Shigella spp. Methods The virulence genes stx1, stx2, eaeA, lt, stIb, aggR, ipaH as the target genes and 16S RNA gene rrs as an internal control gene were grouped into a multiplex PCR reaction system. By optimizing the multiplex PCR primers, reaction conditions and cycle parameters, the multiplex PCR can identify and differentiate the five main pathotypes of diarrheogenic Escherichia coli and Shigella spp rapidly and accurately. Results The established multiplex PCR assay was applied in this study to identify 87 strains of Escherichia coli and Shigella spp. The results showed that the genetypes were 100% consistent with those of single PCR amplification. Conclusion The multiplex PCR assay in this study can be used not only to identify the five main pathotypes of diarrheogenic Escherichia coli and Shigella spp in a single reaction, but also to evaluate the efficiency. This assay is an effective supplement to other molecular Methods in the identification of the five main pathotypes of diarrheogenic Escherichia coli and Shigella spp.