Ziziphora clinopodioides (Lamiaceae) is recognized as a native Iranian herb used for the treatment of diarrhea, intestinal gas, nausea, and vomiting. There is lack of consensus among some authors on its taxonomic boundaries at the subspecific level. To resolve this issue, the present study aimed to employ a combination of morphological, molecular, and phytochemical analyses. Firstly, 90 vegetative and floral characteristics were measured from 90 specimens of this species followed by conducting multivariate analyses on the 46 significantly discriminating morphological traits. Additionally, molecular markers (SRAP) were utilized generating 601 total loci of which 593 (98.31%) were polymorphic. The SRAP markers proved to be efficient, as demonstrated by the principal component analysis plot, which grouped all individuals according to their morphotypes, regardless of geographical location. According to the analysis of molecular variance (AMOVA), 10.69% of the total variations were associated with within-populations, while 89.31% were observed among populations. Furthermore, chemical analysis of essential oils (EOs) revealed the presence of Pulegone, 1,8-Cineole, and P-Mentha-3-n-8-ol as the major (>10%) EOs constituents in Z. clinopodioides. Notably, eight EOs compounds (Menthone, B-Pinene, Terpinen-4-ol, Minth-furanone, a-Thujene, Tricyclene, Caryophyllene-oxide, and Germacrene-d) identified in this study, show reliable yields with chemotaxonomic significance within the complex. A high correlation between the genetic markers and EOs content (Pearson’s correlation coefficient, r = 0.6) suggests that genetic markers may be linked to the EOs constituents. Overall, our results obtained from a combination of morphological, molecular, and chemical analyses indicate that nine subspecies of the Z. clinopodioides complex could be decreased to four subspecies

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