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<i>Pyrenophora graminea</i> is the seed-borne pathogen causal agent of barley leaf stripe disease. In this work, we screened a collection of 206 spring two-row barley cultivars from Europe for their resistance to the fungal pathogen. Artificial inoculation with the highly virulent isolate Dg2 revealed a continuous variation for the incidence of infection, with few highly resistant or highly susceptible genotypes. On average, old cultivars showed higher resistance than the more modern ones. Genome-Wide Association Scan was performed by exploiting available molecular data for >4000 SNP markers and revealed a single, highly significant association on the short arm of chromosome 6H, in a genomic position where quantitative trait loci (QTL) for barley resistance to <i>P. graminea</i> were not detected before. Based on the last version of the reference barley genome, genes encoding for proteins with a kinase domain were suggested as candidates for the locus.