Background and Objective: Dimocarpus longan has been known widely due to its many nutritional values for human health. Therefore, this study aimed to evaluate genetic diversity in the matK gene of D. longan varieties in the Mekong Delta. Materials and Methods: Eleven D. longan varieties were extracted by modified CTAB procedure and amplified matK gene by matK-4600/trnK-2R and tamF/tamR primers. The DNA sequences were treated by BioEdit 7.0, MEGA 11, Geneious 7.0, Denovo, ClustalW, NCBI/BLAST tool and compared with D. longan-NC_037447.1 on NCBI. The phylogenetic tree was analyzed by the maximum-likelihood method. Results: The results successfully amplified the matK gene with a size of ~1500 bp. The longan varieties had 3 SNPs that were respectively updated in GenBank (NCBI) with accession numbers including OP819669, OP819670, OP819671, OP819672, OP819673, OP819674 and OP819676. Compared with D. longan-NC_037447.1, all sequences showed similarity ranging from 99-100%. The results of the phylogenetic tree analysis showed that the bootstrap values were >75% in all branches. Conclusion: The sequences of the matK gene were relatively phylogenetically distinguishable between the longan varieties of the Dimocarpus genus.