Fusarium head blight (FHB), is mainly caused by various Fusarium spp. of which the most important is Fusarium graminearum. FHB is considered as the most serious disease affecting wheat (Triticum aestivum L.), causing significant yield losses worldwide. Yield losses result by reduction in grain quality due to shriveled and/or discolored kernels, which are referred to as Fusarium-damaged kernels (FDK) and contamination of grain with the trichothecene mycotoxin deoxynivalenol (DON).In this project, we performed transcriptome analysis of the resistant cultivar AC Morley, AC Emerson and CDC Falcon. AC Morley (Siouxland/ Perlo) is a high yielding and widely grown eastern Canadian winter wheat variety that is moderately resistant to FHB, and Emerson(McClintock/CDC Osprey) is a high yielding winter wheat from western Canada that is rated “R” (or Resistant) for FHB. CDC Falcon is susceptible to FHB. Spikelets and rachis of infected wheat spikes were collected at different days post inoculation and subjected to histological examination by confocal and fluorescence microscopy. The comparative infection pattern helped us to determine the best time point and spike tissue types to be collected for further RNA-seq analysis. Spike tissues collected seven dpi, as well as tissues from the noninoculated spikes were used for RNA extraction. Multiplexed cDNA libraries were prepared and sequenced using the Illumina HiSeq 2500 platform. Transcriptome analysis detected up-regulation of defense signaling genes, mycotoxin detoxification, pathways involve in plant defense response, disease resistant genes and pathogenesis-related proteins. Potential F. graminearum target pathogenicity factors (target genes) were selected based on their differential expression and association with enriched GO terms. The transcriptomic analysis of these winter wheat cultivars using RNA-seq will help us understand their molecular mechanism of FHB resistance.