AbstractPowdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive diseases that pose a great threat to wheat production. Wheat landraces represent a rich source of powdery mildew resistance. Here, we report the map-based cloning of powdery mildew resistance gene Pm24 from Chinese wheat landrace Hulutou. It encodes a tandem kinase protein (TKP) with putative kinase-pseudokinase domains, designated WHEAT TANDEM KINASE 3 (WTK3). The resistance function of Pm24 was validated by transgenic assay, independent mutants, and allelic association analyses. Haplotype analysis revealed that a rare 6-bp natural deletion of lysine-glycine codons, endemic to wheat landraces of Shaanxi Province, China, in the kinase I domain (Kin I) of WTK3 is critical for the resistance function. Transgenic assay of WTK3 chimeric variants revealed that only the specific two amino acid deletion, rather than any of the single or more amino acid deletions, in the Kin I of WTK3 is responsible for gaining the resistance function of WTK3 against the Bgt fungus.