The country of Iran has valuable genetic resources of medicinal plants. Yarrow ( Achillea ) belongs to Anthemideae subfamily and Asteraceae family possesses more than 100 species in the world. Nineteen Achillea species were distributed in different parts of Iran. These species have different pharmaceutical properties and they can be also used for landscaping purposes in arid environments. Drought is one of the physical stresses and considered as one of the most limiting factors of plant growth and production in Iran and some other countries of the world. Environmental stresses can cause metabolism disorders in plant cells which lead to increase Reactive Oxygen Species (ROS). Plants can decrease the destructive effect of ROS by using enzymatic and non-enzymatic antioxidants. Furthermore, accumulation of free radicals can elevate flavonoids and phenolics content in plant cells, because of their considerable antioxidant activity. In the present study, genetic variation for drought tolerance and secondary metabolite in 20 accessions belonging to seven Achillea species ( A. millefolium , A. pachycephala , A. aucherii , A. tenuifolia , A. kellalensis, A. filipendulina and A. nobilis ) were evaluated under 4 different irrigation regimes. The study was performed as a factorial experiment (20×4) in a randomized complete block design (RCBD) with 3 replications for 2 years in green house. The results revealed that A . pachycephala accession from Salafchegan possessed the highest phenolic, flavonoid content and antioxidant activity in both stress and non-stress conditions, while A. kellalensis and A. nobilis accessions had the lowest amount of these compounds. Furthermore , A. pachycephala and A. aucherii showed more tolerance to drought stress in comparing to the other accessio therefore, for molecular studies the species of A. pachycephala was used. In molecular study, eight genes relating to enzymatic antioxidants ( apx1, gpx1, cat2, dhar, mdar, fe-sod, gr1 and aox1) and seven genes involving flavonoid synthesis ( chi, chs1, pal2, f3h1, f3'h, f3'5'h and fls1 ) were detected. The conserved sequences of these genes were used for primer designing and the amplified fragments in A. pachycephala were sequenced. Then, the expression of the genes was evaluated in stress condition at seven days intervals using Real time PCR. Moreover, in order to evaluate the product of the genes in stress condition, antioxidant enzyme activity was measured. Furthermore, the amount of relevant phenolic acid and flavonoids were determined by High Performance Liquid Chromatography (HPLC). In the initial days of drought stress, the plants increased gene expression of antioxidant enzymes for plant defense. In the seventh day of stress, the concentration of hydrogen peroxide was elevated in compared with control. In the day of 14 and 21th of stress, gene expression of enzymatic antioxidants was also increased and in the day of 28, the highest expression of these genes along with the highest amount of hydrogen peroxide was observed. The HPLC analysis determined 10 major compounds in studied genotype. In overall, the concentration of the most important phenolic acids and flavonoids such as chlorogenic acid, p-coumaric acid, rutin, luteolin-7-o glycoside, 1,3 dicaffeoylquinic acid, luteolin, apigenin and kaempferol were elevated in the day of 21 in compared with control, while they were decreased in the day of 28, in spite of the highest gene expression of phenyl propanoids pathway genes in this day. Therefore, in 21 days of drought stress, the change of gene expression and activation of the primary enzymatic defense system can be used for defeating the oxidative stress. The duration of drought stress can lead to use the secondary antioxidant system (non enzymatic antioxidants) by plant. Take into consideration that before severity of the stress, the phenyl propanoid genes were responsible for storage of phenolic acids and flavonoids; however by activation of secondary defense system, theses important medicinal compounds with high antioxidant activity were responsible to defeat the ROS. Therefore, these compounds were oxidized and their amount was decreased at the day of 28 of drought stress