Wheat powdery mildew is a significant threat to global wheat production, and wild relatives of wheat, such as *Aegilops longissima*, offer valuable sources of resistance genes. This study reports the map-based cloning of the powdery mildew resistance gene *Pm13* from *A. longissima*. *Pm13* encodes a mixed lineage kinase domain-like (MLKL) protein with an N-terminal MLKL_NTD domain and a C-terminal serine/threonine kinase (STK) domain. The resistance function of *Pm13* was validated through mutagenesis, gene silencing, transgenic assays, and allelic association analyses. The development of introgression lines with reduced chromosome segments of *Ae. longissima* containing *Pm13* enables widespread deployment of this gene in wheat cultivars. The cloning of *Pm13* provides insights into the molecular mechanisms underlying powdery mildew resistance and highlights the role of kinase fusion proteins (KFPs) in wheat immunity. The study also describes the subcellular localization, structural analysis, and phylogenetic analysis of the Pm13 protein, further elucidating its unique domain architecture and evolutionary relationships.Wheat powdery mildew is a significant threat to global wheat production, and wild relatives of wheat, such as *Aegilops longissima*, offer valuable sources of resistance genes. This study reports the map-based cloning of the powdery mildew resistance gene *Pm13* from *A. longissima*. *Pm13* encodes a mixed lineage kinase domain-like (MLKL) protein with an N-terminal MLKL_NTD domain and a C-terminal serine/threonine kinase (STK) domain. The resistance function of *Pm13* was validated through mutagenesis, gene silencing, transgenic assays, and allelic association analyses. The development of introgression lines with reduced chromosome segments of *Ae. longissima* containing *Pm13* enables widespread deployment of this gene in wheat cultivars. The cloning of *Pm13* provides insights into the molecular mechanisms underlying powdery mildew resistance and highlights the role of kinase fusion proteins (KFPs) in wheat immunity. The study also describes the subcellular localization, structural analysis, and phylogenetic analysis of the Pm13 protein, further elucidating its unique domain architecture and evolutionary relationships.