We characterized three lesion mimic necS1 (necrotic Steptoe) mutants, induced by fast neutron (FN) treatment of barley cultivar Steptoe. The three mutants are recessive and allelic. When infected with Puccinia graminis f. sp. tritici pathotypes MCC and QCC and P. graminis f. sp. secalis isolate 92-MN-90, all three mutants exhibited enhanced resistance compared to parent cultivar Steptoe. These results suggested that the lesion mimic mutants carry broad-spectrum resistance to stem rust. In order to identify the mutated gene responsible for the phenotype, transcript-based cloning was used. Two genes, represented by three Barley1 probesets (Contig4211_at and Contig4212_s_at, representing the same gene, and Contig10850_s_at), were deleted in all three mutants. Genetic analysis suggested that the lesion mimic phenotype was due to a mutation in one or both of these genes, named NecS1. Consistent with the increased disease resistance, all three mutants constitutively accumulated elevated transcript levels of pathogenesis-related (PR) genes. Barley stripe mosaic virus (BSMV) has been developed as a virus-induced gene-silencing (VIGS) vector for monocots. We utilized BSMV-VIGS to demonstrate that silencing of the gene represented by Contig4211_at, but not Contig10850_s_at caused the necrotic lesion mimic phenotype on barley seedling leaves. Therefore, Contig4211_at is a strong candidate for the NecS1 gene, which encodes a cation/proton exchanging protein (HvCAX1).