One of the important activities to achieve drought tolerant cultivars is screening and selection of cultivars based on yield and biochemical characteristics. A split-plot experiment arranged in a randomized complete blocks design with three replications was carried out in Safiabad Agricultural Research Center during two growing seasons (2017-19). Main plots consisted of different irrigation regimes based on crop’s water requirement (25%, 50%, 75% and 100%) and sub plots included five levels of alfalfa cultivars (Baghdadi, Nikshahri, Yazdi, Omid and american Mesa-Sirsa). Results showed that drought stress due to irrigation regimes led to significantly reduce of forage dry matter yield and significantly changes of soluble sugar, glutation peroxidase and catalase in alfalfa leaves but it did not have significantly effect on total protein. For forage dry matter yield, the highest statistical class belonged to Baghdadi cultivar under 100% water requirement supply treatment (2.824 t.ha-1). The lowest statistical class for forage dry matter yield was observed in Yazdi and Mesa-Sirsa cultivars under 25% water requirement supply treatment (0.598 and 0.546 t.ha-1), respectively. Among the cultivars that are examined in this experiment, Yazdi was identified as a drought tolerant cultivar due to the stability of dry forage yield and also high leaf antioxidants content.