Fusarium graminearum, the major species causing the Fusarium Head Blight (FHB) disease in cereals produces a variety of mycotoxins. Among these mycotoxins deoxynivalenol (DON) is a major challenge for quality of malting grains. An increase in DON following the malting of grain that had a relatively low initial level of these neurotoxins, and also had been stored for several months are seen as aberrant behavior. Maltsters have speculated that in barley, this may relate to internal vs external infection with Fusarium species. To track the presence and progression of Fusarium mycelia distribution during the malting of FHB infected small grains, we optimized and utilized a fluorescent staining method of cross sectioned grain samples using WGA-Alexa Fluor 488 fluorophore which preferentially binds with sialic acid and N-acetylglucosaminyl residues in fungal hyphae. Confocal laser scanning microscopy evaluation revealed that fungal hypha was mainly present in the husk (spongy parenchyma and cementing layer) of barley, but also can be found in pericarp, testa, aleurone layer, and even slightly into the interspace of starchy endosperm. Extensive growth of fungal hyphae was also observed in pericarp, testa, aleurone layer and the central endosperm of rye and triticale, which illustrated internal infection in rye, triticale and their malts. A sharp increase of DON and Tri5 DNA levels were observed following the malting of these grain samples. To investigate the Tri5 gene expression during infection, we are currently employing the RNAscope RNA in situ visualization. We conclude that the presence of internal infection and viable Fusarium hyphae provides a survival advantage to the pathogen during the harsh conditions of the malting process, thus, contributing to elevated DON level beyond the acceptable limits.