Water stress has become among the most limiting factors of productivity and plant development. To survive under these conditions, several plants have developed many strategies aimed at protecting plant functions. The aim of this study is to evaluate water stress effects on three alfalfa (Medicago sativa L.) populations (Chenini, Gannouch, and Tebelbou) cultivated in southern Tunisian oases. To investigate the effect of water stress on alfalfa, four treatment levels were applied: 25, 50, 75, and 100% (control) of field capacity with four replicates for each treatment. Drought application led to a high reduction (p<0.05) on net photosynthetic rate (P_N), stomatal conductance (g_s), water use efficiency (WUE), relative water content (RWC), leaf area (L_A), and chlorophyll a and b content while leading to an increase on water use efficiency of productivity (WUE_p) and on intracellular CO₂ concentration (C_i). Both stomatal and non-stomatal limitations govern photosynthesis variation in Medicago sativa plants. The Tebelbou population seems to be the most tolerant population as compared to the other two populations. This tolerance is due to its capacity to reduce the most (L_A) in addition to maintaining the highest values for (WUE_p), which are considered an adaptative mechanism to drought conditions.