Fusarium Head Blight (FHB) is a significant threat to wheat which impacts global food security. FHB-resistant varieties, such as the Chinese cultivar 'Sumai3', containing the major resistance gene, Fhb1, is crucial for controlling this disease. In this study, we examined gene expression in eight hard red spring wheat (HRSW) lines, under three treatments: non-inoculated, mock-inoculated, and Fusarium-inoculated. RNA sequencing using newly annotated Sumai3 genome sequence revealed over 4000 differentially expressed genes (DEGs) across various comparisons with unique expression patterns observed under different conditions, especially on chromosome 3B near the Fhb1 locus. This suggests their potential roles in FHB defense. Although the exact functions of most of these DEGs are yet unknown, the orthologs function of one of the DEGs upregulated in resistant lines, TRAES.SUM.r1.3DG00524960 reveals that it is a putative disease resistance protein gene, suggesting its potential role in defense mechanisms. These insights will assist guide breeding programs by providing new candidate genes for FHB resistance, with future research focusing on validating these markers that signal expression changes across diverse wheat cultivars. This study emphasizes the value of transcriptomic analysis in advancing FHB resistance in wheat breeding efforts.