Tan spot of wheat, caused by Pyrenophora tritici-re- pentis (Ptr), results in a yield loss through chlorosis and necrosis of healthy leaf tissue. The major objective of this study was to compare gene expression in resistant and susceptible wheat cultivars after infection with Ptr ToxA-producing race 2 and direct infiltration with Ptr ToxA proteins. Greenhouse experiments included ex- posure of the wheat cultivars to pathogen inoculum or direct infiltration of leaf tissue with Ptr-ToxA protein isolate. Samples from the experiments were subjected to RNA sequencing. Results showed that ToxA RNA sequences were first detected in samples collected eight hours after treatments indicating that upon Ptr con- tact with wheat tissue, Ptr started expressing ToxA. The resistant wheat cultivar, in response to Ptr inocu- lum, expressed genes associated with plant resistance responses that were not expressed in the susceptible cultivar; genes of interest included five chitinases, eight transporters, five pathogen-detecting receptors, and multiple classes of signaling factors. Resistant and susceptible wheat cultivars therefore differed in their response in the expression of genes that encode chitin- ases, transporters, wall-associated kinases, permeases, and wound-induced proteins, among others. Plants exposed to Ptr inoculum expressed transcription fac- tors, kinases, receptors, and peroxidases, which are not expressed as highly in the control samples or samples infiltrated with ToxA. Several of the differentially ex- pressed genes between cultivars were found in the Ptr resistance QTLs on chromosomes 1A, 2D, 3B, and 5A. Future studies should elucidate the specific roles these genes play in the wheat response to Ptr.