Local genetic resources could constitute a promising solution to overcome drought stress. Thus, eight (8) durum wheat landraces and one improved variety were assessed for drought tolerance in pots under controlled conditions. Three water treatments were tested: control (100% of the field capacity (FC)), medium (50% FC), and severe (25% FC) stress. The assessment was carried out at the seedling stage to mimic stress during crop set-up. Results showed that increased water stress led to a decrease in biomass and morpho-physiological parameters and an increase in antioxidant enzyme activities. Severe water stress decreased the chlorophyll fluorescence parameters, relative water content, and water potential of the investigated genotypes by 56.45%, 20.58%, 50.18%, and 139.4%, respectively. Besides, the phenolic compounds content increased by 169.2% compared to the control. Catalase (CAT) and Guaiacol Peroxidase (GPX) activities increased at 17 days after treatment for most genotypes except Karim and Hmira. A principal component analysis showed that the most contributed drought tolerance traits were Chlorophyll fluorescence parameters, relative water content, and electrolyte conductivity. UPGMA clustering showed that the landraces Aouija, Biskri, and Hedhba exhibited a higher adaptive response to drought stress treatments indicating that water stress-adaptive traits are included in Tunisian landraces germplasm.