The widespread occurrence of Fusarium head blight (FHB) has raised considerable concerns about the risk of mycotoxins in grain-based food. Deoxynivalenol (DON) is the most common trichothecene, and its production in North America is primarily associated with Fusarium graminearum. DON can be conjugated in different forms and the conjugated forms are less toxic than DON in general. Recent surveys of commercial beers have shown that DON and its conjugate, DON-3-glucoside (D3G) were the frequent contaminants and the main sources of this contamination were brewing grains, including malt and cereal adjuncts. The transformation of DON to D3G occurs from the field through food processing and is closely linked to Type II resistance of host plants against Fusarium infection. That is why investigating the transformation of DON to D3G is essential not only for food safety and regulatory compliance but also for evaluating the efficacy of crop resistance strategies. The goal of our study was to explore the transformation during malting by quantifying DON and D3G and measuring the expression of Fusarium Tri5 gene and barley HvUGT13248 gene, which are related to the transformation. The Fusarium Tri5 gene is responsible for the production of DON by encoding a crucial enzyme in the trichothecene biosynthesis pathway and the HvUGT13248 gene in barley is responsible for the transformation by encoding a barley UDP-glycosyltransferase (UGT) specifically catalyzing the conjugation of DON to D3G. FHB-infected barley samples with three varieties, ‘AAC Synergy,’ ‘Conlon,’ and ‘S2M184’ (St. Paul, and Crookston MN) were used. In all samples, DON and D3G were considerably washed off during steeping but reproduced during germination. The DON and D3G level decreased significantly after steeping by an average of 80% and 58%, respectively; however, after germination, it increased significantly by an average of 39% and 50%, respectively. Compared with the germinated barley without FHB infection (control), the HvUGT13248 gene expression was not significantly regulated by steeping, but it was significantly upregulated by germination. Fusarium Tri5 gene expression was upregulated by either steeping or germination varied by varieties.