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Powdery mildew, caused by Blumeria graminis f. sp. hordei (Bgh), severely impacts global barley Hordeum vulgare L. (Hv) production. This investigation evaluated Egyptian barley genotypes to identify novel resistance sources and molecular markers for breeding programs. Phenotypic assessments at the seedling (growth stage, GS 32) and adult plant (GS 55–59) stages under controlled and field conditions, combined with SSR marker analysis, revealed distinct resistance profiles. Genotypes Giza 123, Giza 125, and G8 exhibited strong resistance, with Giza 123 displaying Mlo-mediated immunity. Susceptible genotypes (Giza 126, G1, G2, and G4) showed rapid disease progression (IT4; up to 80% severity). Intermediate genotypes (G5, G6, and G9) suggested quantitative resistance. Simple sequence repeat (SSR) analysis linked the EBmac0603 primer 160 bp allele to resistance and the 149 bp allele to susceptibility. The EBmac0603 primer 185 bp allele correlated with partial resistance, highlighting its utility in marker-assisted selection (MAS). The integration of phenotypic and molecular data identified Giza 123 and G8 as prime candidates for breeding, emphasizing the need for strategies like gene pyramiding or quantitative resistance incorporation in susceptible lines. This study underscores the value of Egypt’s barley diversity in advancing durable disease resistance through targeted breeding and molecular tools.