Durian (Durio zibethinus) is one of the special fruits in Vietnam favored with high trade in the world. Currently, there are many varieties of durians grown in the Mekong Delta, and it is challenged to distinguish them through morphology. This study was aimed to examine genetic diversity based on DNA barcode and the ISSR molecular marker. The DNA sequences of three barcode DNA candidates, including ITS, matK, rpoC1 of nine varieties (Ri-6, Monthong, Kho Qua Xanh, Chin Hoa, Sua Hat Lep, Chuong Bo, Bi, Musang King, and Sau Huu) collected from Can Tho, Tien Giang, Ben Tre and Vinh Long were sequenced and analyzed. Six SNPs were identified from the ITS sequence between individuals Ri-6-Ben Tre, Monthong-Tien Giang, Chuong Bo-Tien Giang, Sua Hat Lep-Can Tho, and Sau Huu-Tien Giang. For the matK gene, nine SNPs were found to distinguish Ri-6 individuals (Can Tho and Southern Fruit Research Institute), Chin Hoa-Ben Tre, Sua Hat Lep-Ben Tre and Sau Huu-Tien Giang. The rpoC1 gene was highly conservative between varieties in this study. The ISSR molecular markers classified such durian varieties into five groups and showed clear differences between the exotic varieties of Musang King-Vinh Long and Monthong-Tien Giang.