Fusarium Head Blight (FHB) is a globally destructive disease that affects small grains, causing significant yield and quality losses. South Dakota ranks as the 12th barley-producing state in the US. FHB is one of the major diseases that have significantly reduced barley production in the Midwestern United States. However, the recent reports of the Brewers Association suggest a six percent dollar growth in the market share of craft beer, which has escalated the demand for barley within the brewing industry. This shift has placed significant emphasis on developing FHB-resistant barley cultivars with reduced accumulation of mycotoxins, to meet the stringent requirements of the brewing industry. In this comprehensive study, a set of 29 spring barley genotypes, sourced from public and private breeding programs across the United States, underwent screening for FHB resistance. A comparative analysis of key factors, including disease severity, Fusarium Damaged Kernels, and accumulation of mycotoxin content, was conducted across these diverse barley genotypes in two consecutive years, 2022 and 2023. This research revealed substantial variability in disease severity and mycotoxin content. Most genotypes exhibited a positive correlation between the causal fungal pathogen, Fusarium graminearum incidence, and the accumulation of mycotoxins, particularly deoxynivalenol. Specifically, statistical tests indicated that the genotype factor significantly influenced disease severity, DON, NIV, FDK, and mycotoxin accumulation in both years. Notably, in both years of field testing 'S2M190' consistently displayed moderate resistance with lower disease severity, fewer FDK, and minimal mycotoxin content. Several other genotypes exhibited low DON levels in either 2022 or 2023. Further, the results can be validated through greenhouse and multi-location trials for effective disease screening. These findings provide valuable insights into genotype performance, with implications for breeding and crop management strategies to mitigate FHB's impact on barley production. The identified superior cultivars, when integrated into appropriate crop rotation practices, hold the potential to significantly contribute to Fusarium head blight (FHB) management.