Selenicereus species is one of the valuable fruiting plants in Vietnam due to its properties, e.g., rich nutrition and medicine activity. Eight DNA barcodes applied to the discrimination power of dragon fruit species in the Mekong delta. Total DNA extracted from fresh roots and the loci of interest were amplified and sequenced. DNA sequences were aligned and determined variable regions. The findings revealed that four loci, including matK, rbcL, rpoC1, and atpF-H reached high PCR yield and specificity compared to those in ycf1b, psbK-I, and ITS. The atpF-H was the most variable region due to the number of single nucleotide polymorphisms (SNPs) and indel mutations, whereas rpoC1 was the least one. Based on sequence characteristics, each locus only discriminated some of the Selenicereus monacanthus from Southern Horticultural Research Institute identified by combining three loci, atpF-H, matK, and rbcL. The results elucidated the close genetic relationship between Mekong delta dragon fruits and National Center for Biotechnology Information (NCBI) database. Furthermore, this finding generated a DNA barcode database of ten dragon fruit accessions and suggested that multiple loci in the chloroplast genome should be a reliable solution for identifying this highly commercial fruiting plant.