Thymus persicus (Ronniger ex Rech. f.) Jalas (Lamiaceae) is an endemic and valuable essential oil-bearing plant. Herein, the effect of ploidy level on both anatomical structure and essential oil composition was investigated. Leaves of in vitro-induced tetraploids had more glandular peltate, capitate, and elongated non-glandular trichomes compared to diploids. Gas chromatography-flame ionization detector (GC-FID), and GC–mass Spectrometry (GC-MS) techniques were employed to unravel chemicals in hydro-distilled essential oils from in vitro diploid, tetraploid, and mixoploid clones. Notably, 21, 27 and 17 compounds representing 97.0%, 96.9%, and 96.7% of the total essential oils were identified and quantified in diploids, tetraploids, and mixoploids plants, respectively. A wide chemical variability was observed so much so that thymol (32.8%), α-terpineol (26.4%), and carvacrol (6.2%) were the main constituent in diploids, while carvacrol (33.85%), δ-selinene (8.79%), ishwarone (8.23%), and octanoic acid (8.07%) were in tetraploids and mixoploids mostly accounted in δ-Selinene (21.26%), pogostol (13.92%), methyl thujate (10.94%), and (2E,6E)-farnesyl acetate (10.28%). To further underline the ploidy effects, it was observed a strong decrease in the contents of monoterpene hydrocarbons and oxygenated monoterpene hydrocarbons in tetraploids and myxoploids. The sesquiterpene hydrocarbons showed an inverse behaviour.
