Fusarium head blight (FHB), predominantly caused by Fusarium graminearum, is a destructive disease that poses a serious threat to wheat (Triticum aestivum L.) production around the world. A suppression subtractive hybridization (SSH) cDNA library was constructed from F. graminearum infected spikes of a resistant Belgian winter wheat variety Centenaire, exhibiting Type II resistance to FHB. Forty-three differentially expressed transcripts were identified and classified in different categories according to their predicted function, including proteins involved in defense response, signaling, transport of molecules, metabolism and proteins with unknown function. Time-course gene expression analysis between the FHB resistant genotype Centenaire and the susceptible genotype Robigus was carried out on twelve selected genes in order to validate the SSH screening. Real-time quantitative polymerase chain reaction showed that the selected transcripts were differentially expressed between the resistant and the susceptible genotype at three-time points (24, 48 and 72 h) after inoculation with the pathogen, and mostly, the transcripts accumulation rates were higher in the FHB-resistant as compared to the susceptible one. Thirty identified differentially expressed loci were mapped on the corresponding wheat chromosomes either by in silico analysis or by PCR-based mapping strategy, and fifteen of these loci were located within or nearby chromosomal regions known to have quantitative trait loci for FHB resistance in winter wheat cultivars. This work emphasizes the differential gene expression between the FHB-resistant winter wheat Centenaire and the susceptible Robigus and highlights the putative genes and mechanism involved in the disease resistance reaction.