The yield of durum wheat extremely reduces in response to salinity stress because of several variations in biochemical, physiological and molecular traits in this unfavorable condition. According to the agro-physiological traits under salinity stress, the most resistant and sensitive cultivars were selected from 10 genotypes of durum wheat over a period of 2 years. Afterwards, the molecular, biochemical, and physiological traits in these two genotypes were evaluated. The seedlings (3–4 leaves) were exposed to salinity through irrigating with 0.5 × Hoagland solution containing 200 mM NaCl until physiological maturity. Principal components analysis for the agronomic characteristics and stress resistance index led to identifying Behrang and Arya as the most tolerant and sensitive genotypes, respectively. In these two genotypes in response to salinity stress, osmolyte contents (proline, total soluble carbohydrates and total soluble proteins) and enzymatic antioxidant defence system activities (ascorbate peroxidase, catalase and guaiacol peroxidase) were much higher in the most tolerant genotype than those of the sensitive cultivar. Moreover, the most tolerant genotype showed less amount of oxidative stress parameters (hydrogen peroxide, electrolyte leakage, malondialdehyde and other aldehydes) than the sensitive one. Pyrroline-5-carboxylate reductase (P5CR) and delta-1-pyrroline-5-carboxylate synthase (P5CS) genes expression increased under salinity stress (considering much higher increase in the most tolerant cultivar). Also, proline content was shown to have a significant positive correlation with P5CS and P5CR genes expression levels. Our result not only identified Behrang cultivar as a superior genotype for durum wheat breeding programs, but also represented several efficient mechanisms involved in salt tolerance.