More than 280.000 barley accessions (Hordeum vulgare L.) were recorded in gene banks around the world. Most of the genetic diversity that local or traditional varieties of cultivated crops possess is being lost. The experiment was conducted at the Faculty of Agriculture (Saba Basha), Alexandria University, Egypt, during 2016 to characterize different barley cultivars and lines under different salt concentrations. Fourteen barely cultivars and eight lines were used in the current research. All the tested samples were sown in petri dish (15 cm dimeter) under different salt concentrations (0, 100, 200 and 300 Mm NaCl) using complete randomized design in three replicates. Morphological parameters were selected and measured after two weeks from each replicate. The morphological parameters were germination percentage (%), seedling height (cm) and root length (cm). Grains of each cultivar were squashed and total protein were sequentially separated. One dimensional SDS–polyacrylamide gel electrophoresis was performed to separate the total protein. The genetic relationships among cultivars and lines were measured based on morphological and biochemical markers aiming to use in the future breeding program based on genetic variations. The results revealed significant variations among the tested cultivars and lines under different salt concentrations. Line number 5 and 6 showed the highest mean values of morphological traits under high salt concentrations, while barely cultivars showed different response to salinity levels. The tolerant cultivars showed unique bands in total protein analysis that mean when barely plants subject to abiotic stress such as salinity, plants try to increase the total enzyme contact and some amino acid like proline as defense mechanism for protect the cell wall from damage, this fact was achieved during current results.