<p>Durian (<i>Durio zibethinus</i> Murr.) is a valuable fruit crop in Southeast Asia, with Vietnam emerging as a major exporter. This study aimed to evaluate the genetic diversity of 30 durian genotypes collected from Southern Vietnam using intersimple sequence repeat (ISSR) markers and chloroplast DNA barcoding. Seventeen genotypes were analyzed using 10 ISSR primers, producing 58 loci, of which 44 (75.86%) were polymorphic, indicating moderate genetic diversity (mean PIC = 0.41). Cluster analysis grouped the genotypes into eight distinct clusters, with some showing close relationships, such as Ri6–Monthong and Chuong Bo–Sau Huu (similarity = 0.90). For DNA barcoding, six chloroplast loci were initially targeted; however, only <i>mat</i>K, <i>rbc</i>L, and <i>rpo</i>C1 were successfully amplified and sequenced with the PCR success rate was 87.5%, 100%, and 100% respectively. The <i>rpo</i>C1 region showed the highest variation with haplotype diversity Hd = 0.448 and nucleotide diversity π = 0.093 × 10⁻². In this region, we found one parsimony-informative site at position 506. Sequences from 12 native genotypes and two introduced cultivars were analyzed. Phylogenetic analysis based on concatenated sequences of <i>rbc</i>L<i>–mat</i>K<i>–rpo</i>C1 grouped the genotypes into two main clades, broadly consistent with their morphological classification. Integration of ISSR markers and chloroplast DNA barcoding proved effective in assessing genetic variation and distinguishing between indigenous and introduced cultivars. These findings support germplasm conservation, cultivar authentication, and sustainable durian breeding in Vietnam and globally climate change.</p>