Gene cloning in repeat-rich polyploid genomes remains challenging. Here we describe a strategy for overcoming major bottlenecks in the cloning of the powdery mildew (Pm) resistance gene (R-gene) Pm69 (PmG3M), derived from tetraploid wild emmer wheat. Pm69 is providing resistance to numerous Pm isolates. Conventional map-based cloning approach encountered structural variation that suppressed recombination, while an attempt to sequence isolated 6B chromosome harboring Pm69 failed. Pm69 was finally cloned by anchoring Oxford Nanopore Technology (ONT) contigs to the genetic map, combined with transcriptome sequencing of mutants, identification of a candidate NLR (nucleotide-binding leucine-rich repeat), and validation by virus-induced gene silencing (VIGS) approach. This method enables an efficient dissection of R-gene clusters. The predicted Pm69 protein includes an Rx_N domain with RanGAP interaction sites, NB-ARC and LRR domains. Pm69 is a rare gene located within a rapidly evolving NLR gene cluster. Pm69 was pyramided with stripe rust R-genes Yr15, Yr5, and Yr24 in common wheat lines available for resistance breeding.