The genus Annona includes important commercially grown tropical crops but also has been grown popularly in Vietnam due to its wide range of clinical applications. In this study, two DNA barcodes (rbcL and rpoC1) and two ISSR markers were used to assess the interspecies of eleven Annona samples collected from different provinces in the Mekong Delta. As a result, the rbcL marker was successfully amplified and sequenced for identifying Annona species, while rpoC1 sequences required extensive editing and could not distinguish between A. muricata, A. glabra, and A. squamosa samples in BLASTN analysis. The rbcL and rpoC1 sequences harbor 9 and 7 SNPs, respectively, and showed cross evolution properties of A. squamosal NDL-NB among three species. The phylogenetic analysis revealed that sample NDL-NB, previously recognized as A. squamosa morphologically, was grouped in the cluster of A. muricata based on rbcL sequences, whereas it was distinguished in the clusters of A. squamosal and A. glabra based on rpoC1 sequences. Among 33 bands generated from ISSR primers, 20 bands were polymorphic, accounting for 60.60%. Dendrogram analysis of ISSR data could classify seven amplified DNA samples into three genotypes that were not geographically dependent. In conclusion, the performance of the approved DNA barcodes and ISSR analysis appeared to be species-specific or genus-specific, especially indicating the evolutionary divergence of Annona species derived from a neighboring country and its adaptation to certain geographical locations.