Drought tolerance is a main trait for growing and stabilizing barley productivity in dry areas globally. The current study was conducted to evaluate the morphological and yield-related traits of the barley cultivar “Giza134" in response to drought stress. To determine the impact of drought and stress, the experiment was conducted in the growth chamber and in rainfed conditions. In the growth chamber, seedlings were irrigated, watered (normally), and subjected to 30% PEG-600 (polyethylene glycol 600) as a drought stress condition. Furthermore, barley plants were evaluated during two consecutive seasons, 2021 and 2022, at Nubaria (normal condition), in addition to two different rainfed locations on the northwest coast of Egypt, West Barrani and East Matrouh. Most morphological and yield component traits declined significantly, including plant height (cm), spike length (cm), number of grains per spike, biological yield (BY; ton/fed-1), and grain yield (GY; Ardab Fed-1). Grain yield losses were over 85% in West Barrani and East Matrouh, respectively, compared with Nubaria. To understand the mechanisms of drought tolerance at the molecular level, the gene expression of drought-responsive genes, including HvAPX1 encodes peroxidase, HvFNR encodes ferredoxin-NADP+ reductase, HvDHN1 encodes dehydrin, HvSAM encodes S-adenosyl-L-methionine methyltransferases, HvEDE encodes ER degradation enhancer, and HVABH encodes alpha/beta-hydrolases, were measured in leaf tissues of “Giza 134." The relative expression levels of HvAPX1, HvFNR, and HvDHN1 were significantly (p 0.01) upregulated, with over 8-fold for HvDHN1. while HvSAM, HvEDE and HVABH genes are downregulated in response to drought stress. These findings might provide new insights into the mechanisms of drought tolerance in barley and facilitate future breeding programs for resilient barley crops in a changing global climate.