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Fusarium head blight (FHB) and the associated mycotoxin deoxynivalenol (DON) negatively impact the wheat industry worldwide. In North America, FHB is mainly caused by Fusarium graminearum sensu stricto. The purpose of this study was to evaluate, under storage conditions, the expression of the DON biosynthetic gene trichodiene synthase (Tri5) of F. graminearum in grain of hard red winter wheat cultivars 'Overley' (FHB-susceptible) and 'Overland' (moderately resistant to FHB) from field plots treated or untreated with the triazole fungicide Prosaro and the strobilurin fungicide Headline. Infected grain was stored and periodically sampled to determine gene expression by qRT-PCR analysis. The F. graminearum housekeeping gene GAPDH was consistently detected, indicative of metabolically active fungi, and Tri5 detection was significantly higher in 'Overley' compared with 'Overland'. Tri5 gene expression and DON concentrations showed little to no correlation; consequently, Tri5 expression levels did not accurately predict DON concentrations. The strobilurin did not significantly reduce Tri5 gene expression compared with untreated wheat. In the triazole treatment, a significant reduction in the relative expression of Tri5 was detected after 120 days, as well as a downregulation of Tri5 from 60 to 120 days of storage in 'Overley'. In grain from strobilurin-treated plots of both cultivars, the expression of Tri5 increased from 0 to 30 days after tempering. Genetic expression of Tri5 that is necessary for the production of DON can increase during storage of high-moisture grain. Fusarium fungi can persist in wheat kernels for several months postharvest and may actively produce toxin during this period. U.S. Department of AgricultureUnited States Department of Agriculture (USDA); Secretariat of Higher Education, Science, Technology and Innovation of Ecuador; Nebraska Wheat Board; U.S. Wheat and Barley Scab InitiativeUnited States Department of Agriculture (USDA) [59-0790-7-080, 422288, 422533]; Hatch Multistate Research Capacity Funding Program [1006715] This work was supported by the U.S. Department of Agriculture; The Secretariat of Higher Education, Science, Technology and Innovation of Ecuador; Nebraska Wheat Board; U.S. Wheat and Barley Scab Initiative [59-0790-7-080; #422288; #422533]; Hatch Multistate Research Capacity Funding Program [1006715]. Public domain – authored by a U.S. government employee