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Wheat stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most important foliar disease on wheat in China. Early molecular diagnosis and detection of stripe rust will provide a useful aid to the accurate forecast and seasonal control of this destructive disease. Our objective was to develop PCR assays for the rapid identification and detection of P. striiformis.The genomic DNA of P. striiformis and P. triticina were amplified by a pair of primers derived from conserved β-tubulin gene sequence. A 235-bp specific DNA fragment of P. striiformis was isolated and purified. Based on its sequence, another two primer sets were designed successfully to obtain new sequence-characterized amplified region (SCAR) markers of P. striiformis, which could be amplified in all test isolates of P. striiformis, whereas no DNA fragment was obtained in other nontarget wheat pathogens. The detection limit of the primer set YR (f)/YR (r1) was 2·20 pg μl−1. The new SCAR markers of P. striiformis can also be detected in Pst-infected wheat leaves postinoculated for 2 days.Our assays are significantly faster than the conventional methods used in the identification of P. striiformis.Development of a simple, high-throughput assay kit for the rapid diagnosis and detection of wheat stripe rust would be anticipated in a further study.