Wheat stripe rust, caused by the air-borne fungi Puccinia striiformis f. sp. tritici (Pst), is considered the primary biotic threat to bread wheat production globally. Growing resistant wheat variety is a key method of minimizing the extent of yield losses caused by this disease. Quaiu #3, a common wheat line developed at the International Maize and Wheat Improvement Center (CIMMYT), is immune to stripe rust in Mexico despite exhibiting seedling susceptibility to predominant Pst races. A previous study showed large-effect QTL on the long arm of chromosome 2D that explained 49– 54 % of the phenotypic variation in Quaiu #3 and was designated as adult plant resistance (APR) gene Yr54. It was located in the marker interval of Xgwm301 and wPt-667485. In the present study, we developed 30 STS primers based on the common wheat reference genome sequence of which 10 STS markers co-segregated and mapped 0.6 cM away from Yr54. A physical map of 419 kb from the target gene region contained 12 disease resistance proteins, a transmembrane protein and a cellulose synthaselike protein. Sequence comparisons from chromosome 2D specific contigs of Yr54 mutants and wild type are being used to further delineate candidate gene(s) for Yr54. Cloning of Yr54 will provide more information on the molecular basis of stripe rust APR and diagnostic gene-based tools to facilitate gene combinations that underpin new durable stripe rust resistant wheat varieties.