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This study investigated the effect of drought stress on the amount of phenolic and flavonoid compounds as well as H 2 O 2 and malondialdehyde (MDA) in Achillea pachycephala. The expression patterns of the key genes and their molecular mechanisms in the phenylpropanoid pathway (PAL , CHS , CHI , F3H , F3′H , F3′5′H , FLS) were also assessed during drought stress using quantitative real-time polymerase chain reaction (qRT-PCR). The samples were harvested at 0, 7, 14, 21 and 28 days after exposure to drought stress. High-performance liquid chromatography (HPLC) analysis was performed to determine the changes of phenolic and flavonoid compounds – chlorogenic acid, caffeic acid, rutin, luteolin-7-O-glycoside, 1,3-dicaffeoylquinic acid, apigenin-7-O-glycoside, luteolin, apigenin and kaempferol – during stress conditions. Concentrations of most of the compounds increased with increasing drought stress duration. Most of the phenolic acids continued to accumulate with increasing duration of stress, while flavonoids dramatically decreased at day 28 of stress. Chlorogenic acid was the most abundant phenolic acid (4.97 mg/100 g dry weight [DW]) at the beginning of the experiment, while it decreased at day 7 and increased again at day 21. However, different trends were observed for some flavonoids, such as luteolin and apigenin. At the beginning of stress treatment, high accumulation of free radicals (H 2 O 2) and lipid peroxidation (MDA) led to elevated expression of most of the flavonoid genes. MDA increased from 22.66 to 43.28 μmol g−1 DW at day 28. CHS gene expression was elevated at day 7, while chi gene expression remained unchanged. At the end of the stress period, most of the flavonoid concentrations and expression of the relevant genes also increased. The results can facilitate selection of appropriate drought conditions to obtain the highest levels of flavonoids such as luteolin and apigenin and phenolic compounds such as chlorogenic acid for improved health benefits. Image 1 • The effect of drought on flavonoid genes and metabolites were assessed in Achillea pachycephalla. • The concentration of the phenolics and flavonoids showed different trends in response to drought. • H 2 O 2 and MDA were elevated at the beginning of drought stress. • Most of the genes were upregulated at the beginning and end days of the stress. [ABSTRACT FROM AUTHOR]