<p>Mediterranean cereal crop production is expected to be severely affected by heat waves. Our knowledge on the physiological and molecular responses of barley (<i>Hordeum vulgare</i> L) to elevated temperature is particularly scarce. Here, we performed a multivariate analysis to evaluate the response to heat stress of the most common barley landraces (Arturio, Kounouz, Farandole, Arkansas, Manel, Mercure, Sextine, Imen, Rihane) grown in Tunisia. After an exposure of these genotypes during the vegetative growth stage to high temperature (32&#xa0;°C) for 5 days, measurements of growth parameters along with physiological, biochemical and molecular analyses were performed. Our results showed a large variation among these genotypes in root length, plant height, number of leaves, relative water content (RWC), electrolyte leakage (EL), the contents of chlorophyll, proline, soluble sugars and antioxidants enzymes activities like CAT and SOD. Principal component analysis (PCA) and heatmap analysis showed that Kounouz and other landraces including Imen, Rihane, Manel, and Mercure are predominantly associated with heat stress tolerance parameters like CAT, SOD, RWC, and soluble sugars activities. However, Farandole, Arturio, and Arkansas are rather associated with the oxidative stress marker H₂O₂ and MDA. Kounouz and Farandole appear as contrasting varieties with heat stress tolerant and sensitive traits respectively, as it was confirmed by evaluating the oxidative stress status. Remarkably, expression analysis of stress-responsive genes in the two contrasting genotypes under heat stress, showed higher induction of most of these genes in Kounouz compared to Farandole. Altogether, this study strongly suggests that Kounouz is among the most heat-tolerant genotypes and should be included in future breeding programs for a better adaptation of barley to heat waves.</p>