Medicago truncatula is a forage crop of choice for farmers, and it is a model species for molecular research. The growth and development and subsequent yields are limited by water availability mainly in arid and semi-arid regions. Our study aims to evaluate the morpho-physiological, biochemical and molecular responses to water deficit stress in four lines (TN6.18, JA17, TN1.11 and A10) of M. truncatula. The results showed that the treatment factor explained the majority of the variation for the measured traits. It appeared that the line A10 was the most sensitive and therefore adversely affected by water deficit stress, which reduced its growth and yield parameters, whereas the tolerant line TN6.18 exhibited the highest root biomass production, a significantly higher increase in its total protein and soluble sugar contents, and lower levels of lipid peroxidation with greater cell membrane integrity. The expression analysis of the DREB1B gene using RT-qPCR revealed a tissue-differential expression in the four lines under osmotic stress, with a higher induction rate in roots of TN6.18 and JA17 than in A10 roots, suggesting a key role for DREB1B in water deficit tolerance in M. truncatula.