In Tunisia’s arid and semi-arid lands, drought stress remains the most critical factor limiting agricultural production due to low and irregular precipitation. The situation is even more difficult because of the scarcity of underground water for irrigation and the climate change that has intensified and expanded the aridity. One of the most efficient and sustainable approaches to mitigating drought stress is exploring genotypic variability to screen tolerant genotypes and identify useful tolerance traits. To this end, six Tunisian wheat genotypes (Triticum durum Desf.) were cultivated in the field, under a greenhouse and natural light, to be studied for their differential tolerance to drought stress. Many morpho-physiological and biochemical traits were analyzed, and interrelationships were established. Depending on the genotypes, drought stress significantly decreased plant growth, chlorophyll biosynthesis, and photosynthesis; stimulated osmolyte accumulation and disturbed water relations. The most tolerant genotypes (salim and karim) accumulated more potassium (K) and proline in their shoots, allowing them to maintain better tissue hydration and physiological functioning. The osmotic adjustment (OA), in which potassium and proline play a key role, determines wheat tolerance to drought stress. The calculated drought index (DI), drought susceptible index (DSI), drought tolerance index (DTI), K use efficiency (KUE), and water use efficiency (WUE) discriminated the studied genotypes and confirmed the relative tolerance of salim and karim.