Monitoring the pathogenic fungus of wheat stripe rust, Puccinia striiformis f. sp. tritici, plays a key role in effective control of the disease. In the present study, we developed a specific and sensitive polymerase chain reaction (PCR) assay for detecting the pathogen in wheat (Triticum aestivum) leaves. A pair of primers (PSF and PSR) was designed based on the internal transcribed spacer (ITS) region sequence of P. striiformis f. sp. tritici. PCR products that were amplified with universal primers ITS1 and ITS4 were cloned into pGEM-T Easy vectors and sequenced. The ITS sequence was compared with those of P. striiformis f. sp. tritici, P. triticina, P. graminis f. sp. tritici, Blumeria graminis f. sp. tritici, Fusarium graminearum, Rhizoctonia cerealis, and Bipolaris sorokiniana, which are associated with early symptoms of foliar diseases on wheat. Specificity of the primers was tested in the PCR assays using DNA extracted from all tested P. striiformis f. sp. tritici isolates, other fungal species, and healthy and infected wheat leaves sampled around stripe rust foci in wheat fields, different days after inoculation with P. striiformis f. sp. tritici, as well as asymptomatic wheat leaves sampled around stripe rust foci in the fields. A PCR product of 169 bp was amplified from DNA of all P. striiformis f. sp. tritici isolates. The primers did not amplify DNA from the other tested fungal species. The pathogen was detected from asymptomatic wheat leaves inoculated with P. striiformis f. sp. tritici under greenhouse conditions, as well as leaves sampled around stripe rust foci in wheat fields. Under optimum conditions, the PCR assay was highly sensitive and required only 0.1 pg of the target DNA for a detectable and reliable amplification with the PSF and PSR primers.