Physiological markers may provide a shortcut for identifying drought tolerance in alfalfa (Medicago sativa L.). Thus, the objective of this study was to understand physiological properties of alfalfa to assess drought tolerance of its cultivars. Ten alfalfa cultivars (Qomi, Isfahani, Hamedani, Bami, Ordobadi, Gharayonje, Nikshahri, Yazdi, Baghdadi, and Cody) and four irrigation regimes (55, 65, 75, and 85% depletion of available soil water) were arranged as split plot in a completely randomized block design with three replications to determine the chlorophyll, carotenoid, soluble sugars, proline, relative water contents, and herbage yield response of alfalfa cultivars to drought stress. Physiological and herbage yield traits were drought level‐cultivar‐specific and discriminated the cultivars based on their drought tolerance. Based on the correlation between drought tolerance and measured traits at the highest drought level, carotenoid content (r = 0.94), was the most important marker followed by relative water content (r = 0.92) and soluble sugars content (r = 0.89), respectively, and Baghdadi was the most drought tolerant cultivar. The results showed that the physiological markers may be used to identify alfalfa germplasms for drought tolerance.

Alfalfa (Medicago sativa L.) cultivars respond differently to drought stress levels and oxidative injury. This may provide us with an important tool to identify the chemical mechanisms operative in drought tolerant genotypes. The present experiment was conducted to determine catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POX) activities; malondialdehyde (MDA) content; herbage yield; stress susceptibility index (SSI); and drought tolerance index (DTI) of 10 alfalfa cultivars under four irrigation regimes (55, 65, 75 and 85% depletion of available soil water). Drought stress was observed to reduce herbage yield but increase CAT, APX and POX activities as well as MDA content; however, such alterations were cultivar‐drought level‐specific. With a few exceptions, Baghdadi had the highest CAT, POX and APX activities and lowest MDA content, lowest reduction in herbage yield and the lowest SSI and the highest DTI under all irrigation regimes suggesting that it was the most adapted and drought‐tolerant cultivar. Herbage yield had a significant and positive correlation with POX and a significant and negative correlation with MDA under all irrigation regimes indicating that they could be used as markers for selecting more drought‐tolerant cultivars in alfalfa. Catalase and APX also had a significant and positive correlation with herbage yield, thus they could be used as markers for selection of more drought‐tolerant cultivars. The results suggested that a wide variation in drought tolerance exists within alfalfa cultivars, thus selection for more drought tolerance is possible. In addition, chemical markers such as POX, CAT and APX activities and MDA content may be used to assist in selection for more drought tolerance cultivar in this species.