Berseem clover (Trifolium alexandrinum L.) is an important crop in semi-arid regions; its herbage and seed yields are often reduced by water stress. Our objectives were (i) to determine the effect of water stress, applied after a conditioning period, on water relations, proline accumulation and plant dry weight, and (ii) to investigate if some physiological responses differed in varieties of berseem. Five cultivars (Axi, Bigbee, Lilibeo, Sacromonte and Saniros) were grown in a controlled environment, and subjected to four irrigation treatments (T1, T2, T3 and T4 referring to plants irrigated to field capacity every 1, 2, 3 or 4 d, respectively) during a conditioning period (12 d). T1 treatment indicated the well-watered control, whereas T2, T3 and T4 treatments represented the conditioned plants. Leaf water potential (Ψ), osmotic potential (Ψπ), relative water content (RWC), gravimetric soil water content (GSWC) and leaf proline concentration were recorded during the conditioning period and a subsequent water deficit period (3 d) applied at early flowering growth stage. The conditioned plants subjected to subsequent water deficit maintained higher values of Ψ, Ψπ, RWC and GSWC, and lower values of leaf proline concentration. Reductions in parameter values were inversely related to the water stress severity that plants had previously experienced. At the end of the experiment, T1 showed 42%, 58% and 31% lower values for Ψ, Ψπ and RWC, respectively, than those of T4. Conditioned plants were also shorter and accumulated less leaf, stem and total dry weight. The conditioning treatments did not affect the relation between Ψ and Ψπ since conditioned plants show similar values of Ψπ as the control at the same Ψ value. Thus, drought acclimation in berseem clover contributed to water stress tolerance by the maintenance of tissue hydration. The berseem cultivars examined showed differences in plant growth parameters, but they were very similar for physiological responses to water deficit. The main genetic difference was recorded for turgor maintenance capacity.