Using multiplex real-time PCR, 960 fecal samples collected from poultry, cattle, and diarrheic patients in Bangladesh were screened for diarrheagenic Escherichia coli (DEC). Invasion-related gene virB had the highest prevalence in human patients (41%) and was shown to be positively correlated first with afaB for diffuse adhesion and second with aggR for aggregative adhesion. These three genes were specific to patients. In contrast, Shiga toxin genes stx1 (57%) and stx2 (40%) were prevalent in cattle samples. eae for attaching and effacing lesion, and elt and est of enterotoxins were detected from all three sample sources. Heat map and hierarchical clustering assigned the samples into five different clusters with patient samples positive for virB and afaB placing together in one cluster. Although detection of virulence genes cannot be a direct indication of the distribution of DEC organisms, their detection suggests that Shigella spp. or enteroinvasive E. coli are the most prevalent among diarrheagenic bacteria in Bangladesh and that diffusely adherent E. coli are concomitant bacterium. eae-possessing organisms in patients may come from cattle and poultry sources. The small number of stx-positive patients could be explained by the small number of animal samples that were positive for both eae and stx.