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<i>Cochliobolus sativus</i> (Anamorph: <i>Bipolaris sorokiniana</i>) is the causal agent of several important diseases, including spot blotch, common root rot, and black point, in cereal crops. Three pathotypes (0, 1, and 2) with differential virulence patterns on three barley genotypes (Bowman, ND5883 and NDB112) were identified. The pathotype 2 isolates showed high virulence on barley cv. Bowman but exhibited low virulence on the other two barley genotypes. Our previous studies indicated that the high virulence of the <i>C. sativus </i>pathotype 2 isolate ND90Pr was controlled by a single locus <i>VHv1</i> and genes for nonribosomal peptide synthetases (NRPSs) and/or polyketide synthases (PKSs) were involved in the biosynthesis of the unknown virulence factor. In this study, we identified a genomic region (approximately 137 kb in size) unique to ND90Pr based on genome sequence analysis in comparison with non-pathotype 2 isolates of <i>C. sativus</i>. This unique region contains an AFLP marker co-segregating with <i>VHv1</i> and thus is the location of the gene(s) conferring high virulence on Bowman. Of 43 predicted genes in this region, 17 had predicted annotations including two genes encoding for nonribosomal peptide synthetases (NRPSs). One of the genes for NRPSs was knocked out and the knockout mutants were significantly reduced in virulence on cv. Bowman, suggesting that it is involved in high virulence of the pathogen. Molecular and functional characterization of the genes related to virulence in this region will be presented. <p><p>Keywords: Fungus, Cereals-Grains