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Loose smut (LS) disease is a serious problem that affects barley yield. Breeding of resistant cultivars and identifying new genes controlling LS has received very little attention. Therefore, it is important to understand the genetic basis of LS control in order to genetically improve LS resistance. To address this challenge, a set of 57 highly diverse barley genotypes were inoculated with Egyptian loose smut race(s) and the infected seeds/plants were evaluated in two growing seasons. Loose smut resistance (%) was scored on each genotype. High genetic variation was found among all tested genotypes indicating considerable differences in LS resistance that can be used for breeding. The broad-sense heritability (H 2 ) of LS (0.95) was found. Moreover, genotyping-by-sequencing (GBS) was performed on all genotypes and generated in 16,966 SNP markers which were used for genetic association analysis using single-marker analysis. The analysis identified 27 significant SNPs distributed across all seven chromosomes that were associated with LS resistance. One SNP (S6_17854595) was located within the HORVU6Hr1G010050 gene model that encodes a protein kinase domain-containing protein (similar to the Un8 LS resistance gene, which contains two kinase domains). A TaqMan marker (0751D06 F6/R6) for the Un8 gene was tested in the diverse collection. The results indicated that none of the Egyptian genotypes had the Un8 gene. The result of this study provided new information on the genetic control of LS resistance. Moreover, good resistance genotypes were identified and can be used for breeding cultivars with improved resistance to Egyptian LS.