Drought stress is a major limiting factor for crop production in the arid and semi-arid regions. Here, we screened eighty barley (Hordeum vulgare L.) genotypes collected from different geographical locations contrasting in drought stress tolerance and quantified a range of physiological and agronomical indices in glasshouse trails. The experiment was conducted in large soil tanks subjected to drought treatment of eighty barley genotypes at three-leaf stage and gradually brought to severe drought by withholding irrigation for 30 days under glasshouse conditions. Also, root length of the same genotypes was measured from stress-affected plants growing hydroponically. Drought tolerance was scored 30 days after the drought stress commenced based on the degree of the leaf wilting, fresh and dry biomass and relative water content. These characteristics were related to stomatal conductance, stomatal density, residual transpiration and leaf sap Na, K, Cl contents measured in control (irrigated) plants. Responses to drought stress differed significantly among the genotypes. The overall drought tolerance was significantly correlated with relative water content, stomatal conductance and leaf Na + and K + contents. No significant correlations between drought tolerance and root length of 6-day-old seedling, stomatal density, residual transpiration and leaf sap Cl − content were found. Taking together, these results suggest that drought-tolerant genotypes have lower stomatal conductance, and lower water content, Na + , K + and Cl − contents in their tissue under control conditions than the drought-sensitive ones. These traits make them more resilient to the forthcoming drought stress.