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The compositional dependency of the essential oil of Clinopodium menthifolium subsp. menthifolium (Lamiaceae) on ecological conditions has not yet been investigated. In pursuit of this objective, we assessed the quantity and quality of the essential oil in plants from 11 natural populations from the Central Balkans and one cultivated plant. In order to determine the correlations between essential oil variations and environmental conditions, each habitat was characterised by 36 climatic and 19 bioclimatic parameters. Despite inhabiting diverse climatic zones, altitudes, and biogeographical regions, no significant differences were observed in the yield and qualitative and quantitative composition of the essential oils among the analysed plants. All the samples exhibited essential oil yield ≥ 0.5%, V/w, with piperitone epoxide as the major compound. Among the identified compounds, only limonene and (E)-caryophyllene demonstrated dependence on bioclimatic parameters. The bioclimatic parameters which influence the presence of a larger number of compounds are annual temperature range and the precipitation of the wettest quarter. The consistent quantity and quality of the essential oils of C. menthifolium subsp. menthifolium indicate their probable adaptive significance and could serve as chemotaxonomic features of this taxon.