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Spot blotch, caused by Bipolaris sorokiniana, is a growing concern for barley (Hordeum vulgare) production in most humid, temperate growing regions of the world. Under epidemic conditions, this disease can reduce yields significantly. Reduction of kernel plumpness is associated with lower malt extract and consequently grain quality. Enhanced resistance to this disease is a major goal of western Canadian two‐row barley breeding programmes; however, two‐row barley is considered to be more susceptible than six‐row barley. Plant–pathogen interactions observed for Canadian germplasm are polygenic, where quantitative breeding methods could be beneficial for the development of resistant germplasm. A germplasm panel consisting of 200 two‐row, spring barley genotypes with differential reaction to spot blotch was evaluated at Brandon, Manitoba (MB), and Melfort, Saskatchewan (SK), over eight site years. Genome‐wide association study was conducted using a 50k single‐nucleotide polymorphism (SNP) Illumina iSelect genotyping array. The most‐resistant germplasm was found to carry Midwest Six‐rowed Durable Resistant Haplotype (MSDRH) alleles at the loci located on chromosomes 1H (Rcs‐qtl‐1H‐11_10764), 3H (Rcs‐qtl‐3H‐11_10565) and 7H (Rcs‐qtl‐7H‐11_20162, Rcs5 locus), where the 3H locus was found to be the most under‐represented in Canadian germplasm. Additional marker‐trait associations within nurseries were identified on chromosomes 1H, 2H and 4H. Several of the SNP markers identified were found to be polymorphic within the Canadian two‐row germplasm panel and thus could be useful for enhancing spot blotch resistance in two‐row barley for development of resistant cultivars. [ABSTRACT FROM AUTHOR]