Vibrio cholerae is the etiological agent of cholera that has emerged as an endemic disease in different regions of the world in recent years. Traditional microbial culture and microscopy methods are considered to be the best standard for diagnosing V. cholerae infection. These methods, however, delay any available confirmatory answer by days. Molecular methods have the potential to provide sensitive, accurate, and rapid analysis of V. cholerae infection. We have developed a multiplex PCR assay to detect virulence and toxigenic-associated (VTA) genes (ctxA, tcpA, and ompW). To evaluate PCR specificity, additional bacteria from the enterobacteriaceae family (Salmonella typhi, Shigella dysantry, and entrotoxigenic E. coli) and Aeromonas hidrophyla were examined in this study. Specificity tests were evaluated using the genome dilution method. Importantly, the results show that our PCR specificity method represents the best tool for the rapid detection of VTA genes because of its simplicity, cost effectiveness, and accuracy. This multiplex PCR method can be used for examining the existence of VTA genes in patient samples, and therefore will distinguish V. cholerae from other vibrios and bacteria. This method is able to detect 10-100 colony forming units (CFUs) of V.Cholerae and 8.5-85 picograms (pg) of genomic DNA. The multiplex PCR method is also more specific and sensitive than other methods, validating it as an appropriate and sensitive tool for detecting the presence of toxigenic and pathogenic V. cholerae.