Catharanthus roseus is a medicinal plant which contains high amount of vincristine and vinblastine, two anticancer compounds. In this study, 12 Catharanthus roseus individuals, which collected from three provinces in Mekong delta, were evaluated for their genetic diversity. Total DNA was extracted from fresh leaves and three DNA barcodes candidates were amplified, sequenced and aligned. On the basis of phylogenetic analysis and genetic distance, three DNA markers indicated the narrow genetic relationship among C. roseus accessions, the difference of flower color were not reflected on phylogenetic groups. The inter-genetic distance of ITS, matK and trnS-G were high. Therefore, C. roseus species were authenticated from Jussiaea repens, another species with similar flower and leaves morphology. Such findings proposed a reliable method for identification of medicinal plants.