Breeding for resistance to stripe rust (caused by Puccinia striiformis f. tritici) is essential for reducing losses in yield and quality in wheat. To identify genes for use in breeding, a biparental population of 186 recombinant inbred lines (RILs) from a cross of the Chinese landrace Mingxian 169 and CIMMYT-derived line P9936 was evaluated in field nurseries either artificially or naturally inoculated in two crop seasons. Each of the RILs and parents was genotyped with the wheat 55K single-nucleotide polymorphism (SNP) ‘Breeders’ array and a genetic linkage map with 8,225 polymorphic SNP markers spanning 3,593.37 centimorgans was constructed. Two major quantitative trait loci (QTL) and two minor QTL were identified. The major QTL QYr.nwafu-3BS.2 and QYr.nwafu-7BL on chromosomes arms 3BS and 7BL were detected in all field locations and explained an average 20.4 and 38.9% of phenotypic variation stripe rust severity, respectively. QYr.nwafu-3BS.2 likely corresponds to the locus Yr30/Sr2 and QYr.nwafu-7BL may be a resistance allele identified previously in CIMMYT germplasm. The other minor QTL had limited individual effects but increased resistance when in combinations with other QTL. Markers linked to QYr.nwafu-7BL were converted to kompetitive allele-specific polymerase chain reaction markers and validated in a panel of wheat accessions. Wheat accessions carrying the same haplotype as P9936 at the identified SNP loci had lower average stripe rust severity than the average severity of all other haplotypes.