Alfalfa (Medicago sativa L.) is the most important forage crap in the world and is widely cultivated especially, in arid and semi-arid regions. Because of it’s widely distribution, alfalfa has developed different morphological, physiological, and biochemical mechanisms across environmental stresses, especially drought. Thus, this experiment was conducted to determine catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POX) activities, hydrogenperoxide, and malondialdehyde (MDA) contentsof roots and shoots of ten alfalfa cultivars (Qomi, Isfahani, Hamedani, Bami, Ordobadi, Gharayonje, Nikshahri, Yazdi, Baghdadi and Cody) under sixwater potential (0, -0.2, -0.4, -0.6, -0.8 and -1MP) during germination.Drought stress increased CAT, APX and POX activities, hydrogen peroxide, and MDA contents, in both root and shoot, however such alterations were cultivar-drought level-specific. According to the correlation between measured traits at drought condition, antioxidant enzymes activity in roots and shoots had negative and significant correlation with hydrogen peroxide and MDA contents of both root and shoot. Cultivars with higher enzyme activities had lower hydrogen peroxide and MDA contents (Qomi, Baghdadi, Nikshahri and Bami), while cultivars with lower enzyme activities and higher hydrogen peroxide and MDA content (Isfahani, Ordobadi and Hamedani), suggesting that tolerant cultivars may have retained sufficient water and had active enzymatic defense systems against oxidative injury, moreover, antioxidant enzymes activity can be used as biochemical indicator for drought tolerance in alfalfa cultivars.