Seven barley accessions were selected based on their morphological and agronomic diversity in a randomized complete design with three replications to examine the impact of drought stress on germination of barley cultivars. Seven Egyptian barley varieties, designated as Giza126 (G1), Giza127 (G2), Giza 128 (G3), Giza130 (G4), Giza2000 (G5), Giza133 (G6), and Giza134 (G7), were the initial factors in the controlled experiment. Two irrigation treatments using PEG (6000) solutions at concentrations of 50% and 56 %, respectively, were administered as the second factor, while the third treatment was a simple water control. The effects of genotype, PEG-induced osmotic potentials, and their interactions all considerably changed the examined parameters. Osmotic potential decreased seed germination and growth metrics; however, significant genotypic differences were found. The best cultivars in terms of yield parameters were G3, G5, and G6, which showed tolerance to drought stress, while G1, G2, and G4, G7 cultivars showed less tolerance. Genetic diversity across barley types was investigated using ISSR and SDS-PAGE. Seven barley cultivars from various origins were chosen in order to maximise genome coverage, and they were examined using six chosen ISSR markers. There were 63 amplified bands altogether, 30 polymorphic allelic variants (ranging from 3 to 8 per locus with an average of 5) and 33 monomorphic allelic variations (ranging from 4 to 7 per locus with an average of 5.5) that were found. The calculated genetic distance using ISSR data ranged from 0.813 to 0.972. The two groupings on the dendrogram based on genetic distance estimation are typically congruent with the currently available genetic data. The fact that they had a common ancestor and were grouped together further supported their genetic link. The patterns of SDS-protein banding showed a range of variance that revealed a total of 18 bands with various molecular weights, only 14 of them were polymorphic. All varieties displayed a wide range of genetic diversity, as shown by the ISSR and SDS-PAGE results. These data were supported by growth parameters, which we ranked as drought-stress tolerant, and showed them to be excellent candidates for selective breeding for specific traits and enlarging genetic base.