Despite the fact that diarrhaegenic Escherichia coli (DEC) has been identified as a major etiologic agent of childhood diarrhea which represent a major public health problem in developing countries, only a few studies have been performed in Bangladesh to identify these organisms. To detect DEC in patients with acute diarrhea, a total of 300 stool specimens were tested by multiplex polymerase chain reaction (PCR). The multiplex PCR was designed for the detection of target genes of "eae" for enteropathogenic E. coli (EPEC), "stx" for enterohemorrhagic E. coli (EHEC), "ipaH" for enteroinvasive E. coli (EIEC), "aspU", "CVD432" and "aggR" for enteroaggregative E. coli (EAEC) as well as "elt" and "est" for enterotoxigenic E. coli (ETEC). Out of 300 stool specimens collected from patients with acute diarrhea, the DEC was detected in 18% (54/300) cases. The dominating strain was ETEC (13%, 39/300), followed by EAEC (5%, 15/300) and no EHEC, EIEC and EPEC could be detected. Both heat-stable toxin (ST) and heat-labile toxin (LT) genes of ETEC were detected in 66.68% (26/39) strains and only ST or LT as single gene was detected in 23.07% (9/39) or 10.25% (4/39) strains respectively. The multiplex PCR assay could be used as a rapid as well as efficient diagnostic tool for identification of DEC in the clinical laboratory settings.