Background: Thymus daenensis is on e of the endemic aromatic species in Iran that its essential oil is used in food and pharmaceutical industries. Objective: In this study, the effects of plant growth stages and distillation methods on essential oil content and thymol percentage of this species were evaluated. Methods: The aerial parts of Th. daenensis were collected in three stages of plant growth from Tehran province of Iran. The plant materials were dried in shade and their essential oils were isolated by hydro-distillation in three replications. In addition, the essential oils of plant materials at full flowering stage were obtained by hydro-distillation, water&steam distillation and steam distillation. The essential oils were analyzed by capillary GC and GC/MS. Results: The result showed that the different plant growth stages and distillation methods had significant effect on essential oil content. The highest essential oil content was obtained at full flowering stage (2.28% w/w). Among distillation methods, the highest essential oil content was obtained by hydro-distillation (2.27%) that was significant difference with other distillation method. There was no significant difference between essential oil composition at different plant growth stages. Also, thymol, p-cymene and -terpinene were the main components. The highest percentage of thymol was obtained by hydro-distillation. Conclusion: Generally, it can be concluded that harvesting of Th. daenensis at full flowering stage and extraction of its essential oil by hydro-distillation were more suitable for obtaining the higher oil content and thymol percentage.