Fusarium head blight (FHB) poses a significant threat to barley production by reducing both yield and grain quality. Genetic diversity of Fusarium species that cause the disease has potential to impact breeding efforts aimed at increasing FHB resistance of barley. To address this issue, we are assessing genomic variation, virulence, and toxin production of 97 isolates of Fusarium from barley screening nurseries in six U.S. states. Analysis of whole genome sequences revealed that all isolates were Fusarium graminearum but varied in trichothecene chemotypes. Population structure and SNP-based Splitstree analyses of the sequence data indicated that 57 isolates were from the NA1 population of F. graminearum; 30 were from the NA2 population; and 10 isolates were admixtures of the two populations. We examined 34 isolates, representing all six states, for their ability to cause FHB and produce deoxynivalenol (DON) on two barley varieties, AAC Synergy and Pinnacle. The results indicated that over half of the isolates tested were more virulent on Pinnacle than on AAC Synergy. Interestingly, disease severity did not always correlate with DON levels, as some isolates produced high DON levels in planta but showed variable FHB severity. Quantitative PCR revealed that fungal biomass varied in AAC Synergy and Pinnacle. Biomass levels were highly correlated with DON levels and moderately with Area Under Disease Progress Curve (AUDPC), suggesting that higher biomass is linked to increased DON and AUDPC. Metabolomic profiling using liquid chromatography-mass spectrometry combined with principal component analysis highlighted distinct differences between barley varieties and Fusarium isolates, independent of DON. These results highlight the complexity of F. graminearum virulence on barley and indicate that both genetic and environmental factors impact disease expression, providing insight into potential mechanisms to increase FHB resistance in barley.