We analysed the natural variation of drought response in 11 Tunisian populations of Medicago truncatula sampled from environments that varied in soil composition, salinity and water availability. Plants were cultivated in a greenhouse under well-irrigated and water-deficit treatments (50% of field capacity), and a number of traits associated with drought response were measured. Variance analysis indicated that the variation of phenotypic traits was explained by the effects of population, line, treatment, and population × treatment and line × treatment interactions, with treatment being the one with the greatest effect. A large degree of phenotypic variation for most traits in the two water treatments was found within populations. Most of the measured characters showed higher broad-sense heritabilities (H2) in well-irrigated treatment than in water-deficit treatment. Furthermore, the largest population differentiation (QST) for most of the measured traits was observed under drought stress. Most of the correlations between measured traits under both treatments were positive. Four groups of lines differing in drought tolerance were identified, with 45 susceptible, 14 moderately susceptible, 31 tolerant and 23 most tolerant lines. The tolerant group experienced lowest reductions in the length of plagiotropic axes, length of stems, number of internodes and number of leaves. The large phenotypic variation of M. truncatula in response to drought stress can be used to identify genes and alleles important for the complex trait of drought tolerance.