Rambutan (Nephelium lappaceum L.) brings enormous economic value for Vietnam. It is necessary to support the basic research to analyze genetic diversity among rambutan cultivars for identification and conservation. The objectives of this study were to analyze the genetic diversity by DNA barcodes and evaluate polymorphism by ISSR markers on eight rambutan cultivars. Fourteen specimens of eight rambutan cultivars collected to detect the differences in their genotype on the matK and ITS region, combined with ISSR technique. matK gene was sequenced on 12 specimens of rambutan and clustered into three groups on the phylogenetic tree. Besides, nine SNP locations have also been recorded in matK gene and five of them could be used to identify three cultivars including yellow-flesh rambutan, Java rambutan, and the hybrid rambutan Tien Cuong. Results of the polymorphism combining ISSR 10 and ISSR 23 primers gave an average of 11 polymorphic bands per primer. In which, six polymorphic bands were recorded to identify the original rambutan, yellow-flesh rambutan and the hybrid rambutan Tien Cuong. As a conclusion, these diverse characters could be potentially applied in rambutan cultivars identification as well as building a DNA database for Vietnamese rambutan.