Enterohemorrhagic E. coli (EHEC) or Shiga toxin-producing E. coli is known as an important food-borne pathogen, and O157 is the most frequently reported O serogroups of EHEC strains associated with bloody diarrhea and hemolytic-uremic syndrome worldwide. Subsequently O26, O111, O103, O145, O121 and O165 serogroups of EHEC were also frequently isolated from patients with severe diseases in Japan. The O serogrouping of EHEC is essential in outbreak investigations and surveillance. In a previous study, we developed a comprehensive and practical platform for molecular O-serogrouping of E. coli strains, named as E. coli O-genotyping PCR (Iguchi, A., et al .: J. Clin. Microbiol., in press), targeting unique sequences on each O-antigen biosynthesis gene cluster. Based on the PCR system, we now developed an EHEC-specific multiplex PCR system, named as “MP-1 plus,” targeting seven major EHEC O serogroups and three virulence genes, stx1 , stx2 and eae . This primer set contains 10 primer pairs that amplify products with different sizes, and validation studies using reference and wild strains showed that the results were reliable enough. The one-shot PCR method reported here might be a promising tool for the identification and subtyping of EHEC strains for outbreak investigations as well as for the surveillance.