Global food security has been disturbed by global climatic changes and increasing human population around the world. Heat stress is one of most imperative abiotic stress factors that limit barley productivity and production. Herein, three different field screening locations were carried out at Sakha, Mallawi and New-valley research stations, to identify the phenotypic and the genotypic diversity of ten Egyptian barley cultivars during two consecutive seasons 2019/2020 and 2020/2021 under different temperatures degrees. phenotypic diversity was evaluated by using some agro-physiological and grain quality characters which were contributing to yield under heat stress. Genotypic diversity was evaluated by using Sequence related amplified polymorphism (SRAP) markers as molecular identification. The results showed that high temperature enhancement all the cultivars to accelerate flowering by an average (11.39 %) and induce proline content, catalases, peroxidase enzyme active, crude protein content to increasing by average values (79.95, 40.74, 76.42 and 8.20%) respectively. However, it had a negative effect on the remaining characters.Giza 124, Giza 134, Giza 138, and Giza 132 gave high mean performance values of all measured characters being considered as heat tolerance cultivars. Ten SRAP combination primers were used where the percentage of polymorphism for each primer combination varied from 50.0% (me2+em5) to 83.3% (me5+em1). The highest polymorphism information content (PIC) was related to primer me5+em1 (0.399), indicating that this primer is highly informative to be used the barley genetic diversity for heat stress tolerance. A cluster heatmap showed that the ten barley cultivars were clustered into two main clusters, each cluster includes the most closed cultivars together due to their response to heat stress, which could be used as a source in future barley breeding programs for heat stress.