Fusarium head blight (FHB) is a devastating disease in barley, primarily caused by Fusarium graminearum, leading to significant yield loss and mycotoxin contamination, with major economic impacts on the malting and brewing industries. This study hypothesized that variation in Fusarium-related issues during malting may be partially due to differences among F. graminearum strains. Field trials conducted from 2019–2021 used barley cultivars with varying levels of FHB resistance (Newdale and AAC Goldman) inoculated with seven F. graminearum isolates. Disease severity, pathogen density, and deoxynivalenol (DON) content were assessed across years, cultivars, and pathogen strains. Results showed that pathogen strain identity significantly influenced F. graminearum density and DON levels in barley and malt, with Newdale showing higher susceptibility than AAC Goldman. The study also focused on the role of the hydrophobin protein HYD5 from F. graminearum, a surface-active protein associated with fungal pathogenesis and beer gushing. Using a CRISPR-Cas9 system, a ΔHyd5 knockout strain of F. graminearum was generated. The knockout strain showed no significant effect on visual disease severity but resulted in reduced gushing in F. graminearum infected malt. Hyd5 gene expression was elevated during steeping and germination stages of malting, suggesting production of the protein during these phases. Purified HYD5 protein from F. graminearum was sufficient to induce beer gushing, highlighting its potential role in this malting defect. Sequence analysis of HYD5 across Fusarium species revealed interspecies variability, although no intraspecies variation was observed. The findings suggest that managing Fusarium-related malting issues, including beer gushing, requires a deeper understanding of the role of hydrophobins like HYD5 across F. graminearum strains and other Fusarium species that are active in malting environments.