The magnitude of bacterial diarrhea in developing countries is largely unknown because affordable detection methods are not available. We have developed a polymerase chain reaction (PCR)-based assay for use in areas with limited resources to screen for diarrheogenic strains from clinical isolates. To simplify the assay and minimize reagents, our method implemented the use of plasmids rather than bacteria as template controls and the use of bacterial suspensions or crude DNA preparations rather than purified genomic DNA as template DNA. The assay consisted of 3 PCR reactions using 3 groups of 5 to 6 primer pairs to identify the 11 most common bacterial diarrheogenic pathogens. The 3-reaction multiplex PCR amplifies DNA targets specific for each 1 of the 6 Escherichia coli diarrheogenic strains and the 5 non-E. coli diarrheogenic strains, including Salmonella spp., Shigella spp., Campylobacter spp., Yersinia enterocolitica, and Vibrio cholerae. The assay may provide an important epidemiologic tool to investigate the role of diarrheogenic bacterial pathogens in areas of the world with limited resources.