Stripe rust， caused by Puccinia striiformis f. sp. tritici （Pst）， is a global devastating fungal disease that severely affects wheat production. Exploring resistance genes and their use in breeding are the most economical and effective method for controlling stripe rust. Wudubaijianer， a wheat landrace from Longnan region， Gansu province， China， showed moderate to high resistance to stripe rust in the lifecycle， but the resistance basis remains unclear. To decipher its genetic mechanism of stripe rust resistance， the F₂ and F_2：3 populations derived from a cross between Wudubaijianer and a susceptible parent Huixianhong were inoculated with the Pst race V31/lab at the seedling stage， and were sampled for BSE-Seq （Bulked segregant exome capture sequencing）， BSR-Seq （Bulked segregant RNA-Seq） and linkage analysis. The results indicated that resistance to V31/lab in Wudubaijianer was conferred by a recessive locus， tentatively designated yrWUD. Based on BSE-Seq and BSR-Seq， 12 KASP （Kompetitive allele-specific PCR） markers were developed. The linkage analysis mapped yrWUD to a 2.6 cM genetic interval on chromosome 4AL， flanked by markers 4AL36 and 4AL11 with genetic distance of 0.9 and 1.7 cM， respectively， corresponding to a physical interval of 13 Mb （4A：610.26-623.35Mb） harboring three disease-resistance related genes， TraesCS4A02G329100， TraesCS4A02G330000 and TraesCS4A02G330100. These three genes were detected with differential expression between the resistant and susceptible pools， and were proposed as candidate genes for yrWUD. Additionally， the KASP marker 4AL36 was validated in a natural population. Collectively， this study identified a stripe rust resistance locus yrWUD in Wudubaijianer and validated its effect in a natural population， providing a new resistance gene and molecular markers for selecting stripe rust resistance in wheat breeding.