Chloroplast genome sequencing is becoming useful for creating various DNA barcodes. Currently, the utilization of non-coding genes is vital for the systematics and development of flowering plants' plastid DNA barcode. The current work aimed to evaluate the molecular evolution in the almond genus for taxonomic levels and applicability of the two-hotspot barcode plastid coding genes, trnH- and psbA. In this instance, 14 of subg. Amygdalus species of chloroplast genomes were chosen for sequence annotation to look for the highly variable coding DNA barcode sections. Twelve genera were retrieved from the database, while two genera—including the bitter and sweet haplotype—were based on the data of the present study. Results indicated two plastid genes trnH- psbA, with an average length of 1058 bp, has been anticipated to have the higher nucleotide diversity, was the most variable area within the coding genes in the 14 specimens tested. The phylogenetic tree was accurately drawn the monophyletic annotations, providing clear identification without overlap across species among the bitter and sweet almond. The outcomes of data suggest that the sweet almond underwent a domestication happening that originated from the wild almond, peach, and cherry. Whereas the bitter almond was integrated with the wild and cultivated peach indicated its compatibility as the main standard rootstock of peach germplasm. The results obtained here, put forward the domain of the domain of trnH- psbA genes as the most promising coding plastid DNA barcode among the bitter and sweet almond at low taxonomic levels.