Stripe (yellow) rust, caused by Puccinia striiformis Westend. f. sp. tritici Eriks (Pst), is one of the most important wheat (Triticum aestivum L.) diseases and causes significant yield losses. A recombinant inbred (RI) population derived from a cross between Yanzhan 1 and Xichang 76-9 cultivars was evaluated for resistance to wheat stripe rust strain CYR32 at both the seedling and adult plant stages. Four resistance quantitative trait loci (QTLs) were detected in this population, in which the major one, designated as Yrq1, was mapped on chromosome 2DS. The strategy of using the Brachypodium distachyon genome, wheat expressed sequence tags and a draft DNA sequences (scaffolds) of the D-genome (Aegilops tauschii Coss.) for the development of simple sequence repeat (SSR) markers was successfully used to identify 147 SSRs in hexaploid wheat. Of the 19 polymorphic SSRs in the RI population, 17 SSRs were mapped in the homeologous group 2 chromosomes near Yrq1 region and eight SSRs were genetically mapped in the 2.7 cM region of Yrq1, providing abundant DNA markers for fine-mapping of Yrq1 and marker-assisted selection in wheat breeding program. The effectiveness of Yrq1 was validated in an independent population, indicating that this resistance QTL can be successfully transferred into a susceptible cultivar for improvement of stripe rust resistance.