Diarrheal diseases continue to be one of the most common causes ofmorbidity and mortality among young children in developing countries. Theobjective of this study was to assess the usefulness of multiplex PCR as arapid diagnostic tool for simultaneous detection of five categories ofdiarrheagenic Escherichia coli (ETEC, EPEC, EIEC, EHEC and EAEC)using eight virulent genes in four PCR reactions in routine microbiologylaboratory.During the period from August 2011 to March 2012, 140 stoolsamples were collected from children suffering from diarrhea in thegastroenteritis outpatient clinic of Abo El Reesh El Mounira PediatricHospital, Cairo University. E. coli strains were recovered in 100/140 ofdiarrheal samples as identified by colony morphology and conventionalbiochemical tests. We used boiling method for DNA extraction fromE. coli strains. The extracted DNA were used as a template for multiplexPCR.The most common pathotype detected by multiplex PCR was EAEC(59.3%) followed by ETEC and EPEC equally (17.6% each). Four cases ofstx1 gene positive while negative shiga toxin production were also detected.Genotyping characterization of DEC by multiplex PCR will help toclarify the role of E. coli in diarrheal disease, to understand their localepidemiology and to measure the burden of disease in children. If the costand equipment are affordable, characterization of diarrheagenic E .coli bymultiplex PCR is recommended in routine work, otherwise, it could be usedto compare change in prevalence and distribution of different types ofdiarrheagenic E. coli at settled period of time.