Wheat production is constantly threatened by pests and diseases. Leaf rust, caused by the fungus Puccinia triticina, is the most widespread and among the most destructive wheat disease. The introgression of chromosome segments from wild wheat relatives is an attractive strategy to increase diversity of the wheat disease resistance repertoire. Wheat leaf rust resistance genes Lr9 and Lr58 were introgressed into bread wheat from the U genomes of Aegilops umbellulata (2n = 2x = 14, UU) and Aegilops triuncialis (2n = 4x = 28, UtUtCtCt), respectively. Lr9 was mapped to the long arm of chromosome 6B and Lr58 to the long arm of chromosome 2B. To clone Lr9 and Lr58, we developed the MutIsoSeq pipeline, which combines mutagenesis with isoform sequencing to identify candidate genes. Using this approach, we were able to find candidate genes for both Lr9 and Lr58. Surprisingly, the candidate genes for Lr9 and Lr58 have exactly the same coding sequence, translating into a wheat tandem kinase (WTK) followed by a C-terminal von Willebrand factor type A (vWA) domain. We found that Lr9 and Lr58 introgression lines have the same translocation on the long arm of chromosome 6B from Ae. umbellulata. Virus-induced gene silencing of WTK-vWA in the Thatcher-Lr9 and TA5605 backgrounds both resulted in reduced leaf rust resistance. By screening more than 8,000 EMS-induced TA5605 and Thacher-Lr9 M2 families, we found more than 100 leaf rust susceptible mutants, among which, more than 80% of them carry mutations that change the amino acid sequence of WTK-vWA. These amino acids substitution mutations were combined with the WTK-vWA 3D protein structure predicted by AlphaFold to explain the functional mechanism of the WTK-vWA in plant defense. In summary, our work identified an unusual tandem kinase fusion protein conferring leaf rust resistance, expanding the repertoire of disease resistance genes for wheat breeding.