Fusarium graminearum is responsible for Fusarium head blight (FHB), which is a destructive disease of wheat that makes its quality unsuitable for end use. To understand the temporal molecular response against this pathogen, microarray gene expression analysis was carried out at two time points on three wheat genotypes, the spikes of which were infected by Fusarium graminearum. The greatest number of genes was upregulated in Nobeokabouzu-komugi followed by Sumai 3, whereas the minimum expression in Gamenya was at three days after inoculation (dai). In Nobeokabouzu-komugi, high expression of detoxification genes, such as multidrug-resistant protein, multidrug resistance-associated protein, UDP-glycosyltransferase and ABC transporters, in addition to systemic defense-related genes, were identified at the early stage of infection. This early response of the highly-resistant genotype implies a different resistance response from the other resistant genotype, Sumai 3, primarily containing local defense-related genes, such as cell wall defense genes. In Gamenya, the expression of all three functional groups was minimal. The differences in these molecular responses with respect to the time points confirmed the variation in the genotypes. For the first time, we report the nature of gene expression in the FHB-highly resistant cv. Nobeokabouzu-komugi during the disease establishment stage and the possible underlying molecular response.