In order to shed light the transcriptional reprograming of wheat upon Puccinia triticina infection, the wheat near isogenic line TcLr26 (incompatible, resistance) and its recurrent parent Thatcher (Tc) (compatible, susceptibility) were inoculated by physiological race 260, and then harvested for RNA-seq analysis using HiSeq^TM2000 high-throughput sequencing technology. The assembled transcripts were subjected to analyze COG, GO and KEGG classification and functional annotation, pathway annotation and in silico prediction of coding genes. Total 87,291 Unigenes were obtained after de novo assembly, with an average length of 866 bp. The functional clustering analysis revealed 25 COG classifications, 55 GO functional subclasses and 128 KEGG paths. By analyzing the differentially expressed genes (DEGs) between compatible and incompatible combinations, a total of 4,037 up-regulated and 1,949 down-regulated Unigenes were identified at 8 h, and 2,122 up-regulated and 3,248 down-regulated Unigenes were identified at 24 h. The genes, which residue to the calcium signaling pathway, active oxygen burst and SA signaling pathway, were found to be differentially expressed at different time point. The DEGs at 8h and 24h after inoculation were assumed to be closely related to the induction of basic defense response and Lr26-mediated HR defense response, respectively. Taken together, the generated datasets would be useful for further analysis and evaluation of wheat resistance related genes against Puccinia triticina.