AbstractAegilops tauschii (Ae. tauschii) is a diploid (2n = 2x = 14) wild grass species, which has been reported as the progenitor of hexaploid wheat (Triticum aestivum) with D-genome. In this study, 68 Ae. tauschii accessions with diverse geographical backgrounds were investigated for their resistance to infection by the leaf rust fungi Puccinia triticina. Two Ae. tauschii accessions that exhibited hyper-resistance to leaf rust at both seedling and adult stages were identified. Utilizing two susceptible Ae. tauschii ecotypes and keumkang, a common Korean wheat cultivar known to be susceptible to leaf rust, as the negative control, further investigations were conducted for understanding the mechanism underlying immunity to leaf rust disease of these two resistant accessions. Resistant accessions displayed the increased β-1,3-glucanase activity to prevent fungal penetration and the better peroxidase activity to cope with leaf rust-induced oxidative stress. Moreover, transcriptional analyses reveal the important role of the LRR receptor-like serine/threonine-protein kinase FLS2 (lrr) to the disease resistance of the two ecotypes. Ae. tauschii is a remarkable genetic source, especially for abiotic and biotic stress resistance genes, as the plant is known for its wide-ranging geographical habitat and adaptability to different environments. This, combined with the fact that Ae. tauschii and wheat share a close evolutionary relationship, is indicative of the immense benefit of using Ae. tauschii as a material for improving the quality of synthetic wheat. Our aim was to identify and evaluate the strongest Ae. tauschii contenders for breeding leaf rust-resistant synthetic wheat.