Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases of wheat worldwide. To isolate defense-related genes against the pathogen, a suppression subtractive hybridization library was constructed for an incompatible interaction. From the library, 652 sequences were determined to be unigenes, of which 31 were determined as genes involved in signal transduction and 77 were predicted to encode defense-related proteins. Expression patterns of 12 selected signal transduction and defense-related genes were determined using quantitative real-time polymerase chain reaction. Signal transduction genes started increasing their expression at 12 h post inoculation (hpi), and expressions of the most of the transport and resistance-related genes were induced at 18 hpi. The gene expression results indicate specific molecular and cellular activities during the incompatible interaction between wheat and the stripe rust pathogen. In general, the expression increase of wheat signal transduction genes soon after inoculation with the pathogen inducing various defense-related genes, including reactive oxygen species, ATP-binding cassette (ABC) transporters, pathogenesis-related proteins, and genes involved in the phenylpropanoid pathway. The activities of these defense genes work in a sequential and concerted manner to result in a hypersensitive response.