Salinity and osmotic stresses are prime reasons of plant growth and productivity reduction in semi-arid regions and cause complex series of physiological, cellular, and molecular changes. Since osmotic and ionic effects are correlated and initiated by salinity, separating both is an important step in understanding the basis of salt tolerance. Barely seedlings(cultivar Giza 134)were treated with either NaCl(150 mM)or iso-osmotic polyethylene glycol 6000 (19.5% PEG). Treatments were applied two times before sampling and collected after two weeks from emergence. Results showed decreasing of fresh matter in treated seedlings, especially those treated with PEG. Furthermore, significant increase of non-enzymatic antioxidants, oxidative markers in addition to enzymatic antioxidants examined (peroxidase (POX), and polyphenol oxidase (PPO)) was detected with PEG treatment. The osmoregulators including proline (Pro) and glycine betaine(GB) increased in the root tissue, in conjunction with enhancement in the antioxidant status of leaves by applying PEG. Based on molecular analysis using real-time RT-PCR, HvNHX gene (coding for Na+/H+ antiporter) was highly expressed after 48 h from treatment in the roots under salinity, but it was expressed in PEG-treated leaves rather than salt-treated ones, and the opposite was true for HvGORK gene (regulate voltage-gated K+-permeable channels).On the other hand, HvDREB gene(coding for dehydration responsive element binding protein)has recorded higher expression in the roots under PEG treatment compared to control. Taken together, the current study suggests that the studied barley cultivar possesses higher tolerance to salt stress than osmotic stress imposed by PEG, so it could be more suitable for cultivation under salinity conditions.