Alfalfa ( Medicago sativa L.) is the most important forage legume, grown from equator to arctic polar circle areas. Therefore, it has developed different morphological, physiological and biochemical mechanisms to drought stress. Thus, the objectives of this study were to understand morphological, physiological, chemical and biochemical properties of alfalfa cultivars under drought stress, identify morphological, physiological and biochemical markers for drought tolerance, and select drought tolerant cultivar. Three separate experiments including petri dish, pot and field studies were carried out. Sixteen alfalfa cultivars and six water potentials (0, -0.2, -0.4, -0.6, -0.8 and -1 MPa) were arranged as a factorial based on a completely randomized design with four replications. Roots and shoots lengths and fresh weights of alfalfa seedlings were measured. Root and shoot fresh weights and shoot length reduced, while root length increased with increasing drought level, however, there was interaction between drought level and cultivar. Ten cultivars including Qomi, Isfahani, Hamedani, Bami, Ordobadi, Gharayonje, Nikshahri, Yazdi, Baghdadi and Cody were selected for biochemical analysis, pot and field experiments. Hydrogen peroxide and malondialdehid (MDA) contents, and antioxidant enzyme activities increased in the shoots and roots of alfalfa seedlings with decreases in water potential, however, they were cultivar-drought level-specific. Baghdadi had the highest catalase (CAT) activity and Qomi had the highest peroxidase (POX) and ascorbat peroxidase (APX) activities and the lowest hydrogen peroxide and MDA content in the root and shoot. In the pot experiment selected cultivars were evaluated under four irrigation regimes (55, 65, 75 and 85% depletion of available soil water) using a factorial arrangement based on a randomized complete block design with four replications during 2012 and 2013. Root and shoot dry weight, proline, soluble carbohydrates, Ca and K contents, CAT, POX, and APX activities, and MDA content were determined. Shoot height, relative water content (RWC), chlorophyll, carotenoid, protein, acid detergent fiber (ADF) and neutral detergent fiber (NDF) contents, were also measured. Drought stress increased leaf and root proline and soluble sugars contents, shoot carotenoid and protein contents, leaf to stem ratio, leaf and root K, Ca and MDA contents, and root and shoot antioxidant enzyme activities, while, decreased stem height, shoot and root dry weights, chlorophyll content, RWC, ADF and NDF contents. However, the effects were drought level-cultivars-specific and discriminated cultivars based on their drought tolerance. Based on cluster analyses of measured traits, Baghdadi ranked as the most drought tolerant cultivar followed by Qomi, Bami, Nikshahri, Yazdi, Gharayonje, Cody, Isfahani, Ordobadi and Hamedani, respectively. Based on the correlation between stress susceptibility index and measured traits, root soluble carbohydrates (r= -0.95), root proline content (r= -0.95), carotenoid content (r= -0.95), root ascorbate peroxidase activity (-0.95), leaf MDA content (r=0.95) and stem height (-0.93) were identified as the most important markers for drought tolerance in alfalfa cultivars. A split plot based on a completely randomized block design with three replications was also conducted under field conditions using the same irrigation regimes and cultivars during the 2011 and 2012. All of the measured traits were the same as the pot experiment except root traits that were not measured. All of the measured traits were affected by drought level, and cultivar. Baghdadi ranked as the most drought tolerant cultivar. Based on the correlation between stress susceptibility index and measured traits MDA content (r = 0.96), was the most important marker followed by ascorbate peroxidase activity (r = -0.94), carotenoid content (r=-0.91) catalase activity (r = -0.90), respectively. The results indicated that responses of the cultivars to drought level were cultivar-drought level-specific and selection of drought tolerance cultivar based morphological, physiological and bio-chemical markers may be possible. In addition, Baghdadi was the most drought tolerant cultivar and the MDA was the best marker for drought tolerance selection in alfalfa. Key words: Drought stre Alfalfa; Antioxidant; Biochemical markers