Flavonoids accumulate in plants in response to water deficit. Changes in amount, type, and localization of flavonoids under different drought conditions in Arabidopsis thaliana have not been well investigated. Therefore, in this study flavonoid patterns were investigated under water potentials of -0.2, -0.5, and -0.9 MPa at 0, 24, 48, 120, and 192 h after drought induction. Determination of amount and type of flavonoids was performed by HPLC and spectroscopy. In addition, localization of flavonoids was detected by DPBA staining and a fluorescent microscope. Only quercetin and kaempferol were detected in hydrolyzed extracts of roots and shoots. The maximum amounts of the above-mentioned flavonols were detected under severe drought stress. Under all drought conditions, there was more kaempferol than quercetin. Moreover, amounts of both flavonols and total flavonoids were greater in roots than in shoots. Different fluorescence intensities of the flavonoid-DPBA complex were observed in all seedlings from shoots to root tips. The results of this study suggest that flavonoid responses of Arabidopsis to drought stress are dynamic, and intensity and duration of drought stress could play a key role in determination of type, amount, and localization of flavonoids in response to different levels of water deficit.