Mentha longifolia L. is an aromatic medicinal plant species in the Lamiaceae family. This study aimed to assess the diversity of 20 M. longifolia accessions collected from the Zagros Mountain range in Iran and Iraq using morphological and biochemical traits, as well as start codon targeted (SCoT) molecular markers. The evaluation of 24 quantitative and qualitative phenotypic traits revealed considerable variation, particularly for leaf size, stem features and inflorescence morphology. Substantial variation was also found for essential oil content (1.54–5.49%), total phenol content (TPC) (128.59–333.28 mg GAE/g DW), and antioxidant activity (IC50 0.44–0.58 μg/mL). Pearson correlation analysis revealed associations between essential oil percentage and leaf width, leaf number, stem length, and flower number. Principal component analysis (PCA) identified key components of variation including essential oil content, leaf morphology, TPC, and floral traits. SCoT marker analysis generated 86 fragments, with 71.52% polymorphism and an average polymorphism information content (PIC) of 0.34. The lack of geographical clustering among the M. longifolia accessions may be attributed, to some extent, to gene flow or genetic material exchange among populations. The principal coordinate analysis (PCoA) biplot based on the first two PCs further confirmed higher genetic variability among Iranian accessions than the Iraqi counterparts. Notably, the Boukan accession represented a higher genetic distance from other accessions, suggesting that it is genetically unique. The present research revealed significant phenotypic, biochemical, and molecular diversity among M. longifolia accessions from Iran and Iraq. The observed diversity provides opportunities for selecting favorable genotypes. The selected individuals can be used as parents in breeding programs, where controlled crosses and selection combine advantageous alleles to develop new cultivars with improved traits. Accordingly, accessions like Khabat and Harir with high essential oil content and antioxidant potential can contribute novel alleles in breeding programs.
