DNA barcoding is a molecular approach for species identification and germplasm conservation in the Origanum genus. This study proposes a DNA-based method to accurately identify the numerous species within this diverse genus. Four chloroplast genes (matK, rbcL, rpoB, trnL) and a non-coding sequence (psbA) were analyzed. Among the applied barcoding loci, matK exhibited the longest aligned sequence length, followed by rpoB, rbcL, psbA-trnH, and trnL. The rbcL gene showed the highest number of informative sites, while psbA-trnH displayed the highest proportion of variable sites within the Origanum genus. Phylogenetic analysis indicated that rbcL, rpoB, trnL, and psbA sequences were more effective than matK for identifying the Origanum genus, species, and subspecies. Combining multiple sequences improved accuracy. This study provides a molecular reference for herbal product identification and valuable tools for species identification and germplasm conservation within the Lamiaceae family.