The diarrheal disease "cholera" is caused by Vibrio cholerae, and is primarily confined to endemic regions, mostly in Africa and Asia. It is punctuated by outbreaks and creates severe challenges to public health. The disease-causing strains are most-often members of serogroups O1 and O139. PCR-based methods allow rapid diagnosis of these pathogens, including the identification of their biotypes. However, this necessitates the selection of specific target sequences to differentiateeventhe closelyrelatedbiotypesofV.cholerae.Oligonucleotides for selective amplification of smallRNA (sRNA) genes that are specific to these V. cholerae subtypes were designed. The resulting multiplex PCR assay was validated using V. cholerae cultures (i.e., 19 V. cholerae and 22 non-V. cholerae isolates) and spiked stool samples. The validation using V. cholerae cultures and spiked stool suspensions revealed detection limits of 10-100 pg DNA per reaction and 1.5 cells/mL suspension, respectively. The multiplex PCR assay that targets sRNA genes for amplification enables the sensitive and specific detection, aswell as the differentiation ofV.cholerae O1 classical,O1ElTor, andO139 biotypes. Most importantly, the assay enables fast and cheaper diagnosis compared with classic culture-based methods.