<p>Salt stress is one of the major threats affecting crop yield. We assessed the behaviour of three barley genotypes, Ardhaoui, Manel, and Testour under 200&#xa0;mM NaCl with the aim of evaluating the physiological and molecular mechanisms involved in barley salinity tolerance. Results revealed that salinity stress significantly decreases plant growth and water-holding capacity, particularly in the salt-sensitive genotype Testour. Tissue ionic content assessment demonstrated significantly distinct salinity-induced responses. The salt-tolerant genotype Ardhaoui accumulated more K⁺ and less Na⁺ content in both leaves and roots compared with the two other genotypes, leading to an increased K⁺/Na⁺ ratio. Furthermore, the genotype Ardhaoui exhibited a stronger selectivity transport capacity of K⁺ over Na⁺ from root to leaf compared to both Manel and Testour. This effect was due to enhanced K⁺ retention and Na⁺ exclusion, regulated by <i>HvHKT</i> expression. Indeed, higher <i>HvHKT2;1</i> gene transcript abundance was detected in both leaves and roots of the Ardhaoui genotype, as well as an upregulation of <i>HvHKT1;1</i> and <i>HvHKT1</i>, mainly in Ardhaoui roots. In view of the severe impact of salinity on plant development, these findings could be applied to the genetic improvement of plant salinity tolerance.</p>