Barley (Hordeum vulgare L.) is the second cereal consumed and cultivated by the Moroccan population. However, with climate change, it is predicted that frequent drought periods can cause serious plants growth problems. Thus, the selection of drought tolerant barley varieties becomes highly required to ensure the security of barley’s need. Here, we tested drought tolerance of nine barley Moroccan varieties (ADRAR, AMALOU, AMIRA, FIRDAWS, LAANACEUR, MASSINE, OUSSAMA, TAFFA, and TAMELLALT) based on physiological and biochemical parameters. Drought stress was applied by maintaining filed capacity of pots at 40% (90% for the control), and plants were randomly arranged in greenhouse, under 25°C and natural light conditions. Drought stress reduced relative water content (RWC) and chlorophyll content (SPAD index). It also induced a significant increase of electrolytes leakage, hydrogen peroxide, malondialdehyde (MDA), water soluble carbohydrates, and soluble proteins contents. Higher RWC and proline content levels were recorded in FIRDAWS, LAANACEUR, MASSINE, OUSSAMA and TAFFA, which can be interpreted by high drought tolerance. On the other hand, ADRAR, AMALOU, AMIRA, and TAMELLALT showed higher values of MDA and H2O2 contents, which can be linked with drought sensitivity. Physiological tolerance could be a good criterion for selecting barley drought tolerant varieties.