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The effect of water deficit on growth and some physiological and biochemical parameters related to water deficit tolerance was studied in 4 Moroccan alfalfa Medicago sativa L. populations that originated from the mountains and oases of Morocco. The experiment was conducted in greenhouse conditions. Seeds were allowed to germinate in pots filled with sand and peat at a 2: 1 ratio, respectively. After 1 month of sowing, the plants were subjected to 3 water regimes: optimal irrigation (75% of field capacity, FC), moderate water deficit (50% FC), and severe water deficit (25% FC). At 45 days of stress, the plants were harvested and subjected to some agrophysiological and biochemical analyses related to tolerance. Results showed that the water deficit negatively affected dry biomass, membrane permeability, leaves’ relative water contents, and nitrate and phosphorus contents. The behaviors of tested populations were significantly different for most of the considered parameters. The tolerance was positively correlated to the ability of plants to overcome oxidative stress by the induction of antioxidant enzyme activity, accumulation of inorganic ions (Na+ and K+), and maintenance of an adequate level of nitrate reductase and acid phosphatase activities.