In this study, we investigated variation for tolerance to water deficit in 47 lines of Medicago truncatula Gaertn., M. ciliaris (L.) All., and M. polymorpha L. collected from different ecogeographic regions in Tunisia. Plants were cultivated in the greenhouse under 100 and 30% of field capacity and they were harvested at the flowering stage. Nineteen morphophysiological parameters were measured for each species. Our results showed that variation of parameters were explained by the effects of species, line within species, treatment, species × treatment interaction, and treatment × line within species interaction. Drought treatment explained the most variance for the measured parameters. Of the 19 traits, 12 showed significant differences for tolerance to water deficit between M. truncatula, M. ciliaris, and M. polymorpha. M. ciliaris was the latest‐flowering and it exhibits the highest biomass under both treatments. High broad‐sense heritabilities (H2) were noted for most of parameters under control treatment and drought stress. Positive correlations were found between transpiration rate (E) and aerial and root fresh weights, and between photosynthetic rate (A) and stomatal conductance (gs), and between relative water content and number of nodules. Studied lines formed five groups based on drought response indices (DRI). A first group is formed by 23 most tolerant lines, a second group and a fourth group are constituted by 17 moderately affected lines, and a third group and a fifth group have 8 sensitive lines. Tolerant lines of three species can be good candidates in future breeding programs.