Salt stress is one of the most important environmental stresses that negatively affects the plant growth and productivity worldwide. To delineate physio-biochemical and molecular differences among barley genotypes, antioxidant enzyme and transcriptomic profiles along with several physiological traits were compared among a set of elite genotypes of barley subjected to salinity stress. The experiment was conducted at the early growth stage under two control (0 mM NaCl) and salinity (200 mM NaCl) conditions. Salt treatment significantly increased root and shoot Na+ contents, the activity of APX and GPX antioxidant enzymes, and the relative expression of HvHKT2, HvHKT3, HvSOS1, HvSOS3, HvNHX1, HvNHX3, APX, and GPX genes. In contrast, other root and shoot fresh weights, root and shoot dry weights, root and leaf K+ contents, and root and leaf K+:Na+ ratios were significantly decreased due to salinity stress. A principal component analysis showed the relationships among measured traits and relative expression of salt-responsive genes. Remarkably, our results indicated a positive and significant association between the relative expression of HvNHX1 and HvSOS3 genes with root Na+ content and leaf K+:Na+ ratio. Furthermore, associations among relative expression of HvHKT2, HvNXH3, HvSOS1, APX, and GPX genes were positive and significant. Taken together, this study revealed that genotypes G6 responded better to salinity stress than other genotypes. Hence it could be selected as the most salt-tolerant genotype for further complementary experiments with the aim of releasing as a commercial variety. Moreover, this genotype could be used as an ideal parent for transferring favorable physiological and biochemical activities in barley breeding programs.