Fusarium head blight (FHB) is one of the most economically important diseases of wheat and barley because it occurs widely, reduces grain yield and quality, and contaminates grains with mycotoxins. Although efforts to improve FHB resistance in barley are underway at multiple state screening nurseries, it is unclear whether variation in Fusarium strains used by different nurseries impacts efforts to enhance resistance. To address this knowledge gap, we are examining variation in genome sequences, mycotoxin production and aggressiveness of 97 Fusarium isolates from barley screening nurseries in six states. Phylogenetic analyses of DNA sequences from three housekeeping genes retrieved from genome sequences of a subset of 66 isolates confirmed that the isolates were all Fusarium graminearum. Chemical analysis for trichothecene mycotoxin production indicated that nurseries in two states (Idaho and Minnesota) included both 3-acetyldeoxynivalenol (3-ADON)-producing and 15-acetyldeoxynivalenol (15-ADON)-producing isolates, while the nurseries in the other four states included only 15-ADON-producing isolates. One isolate from the Minnesota nursery did not produce any trichothecene mycotoxins in culture. Assessment of disease induced by a subset of 16 isolates on two barley varieties indicated that some isolates differ markedly in aggressiveness. However, most isolates tested caused high levels of FHB. Chemical analysis of both barley varieties following inoculation indicate that DON levels did not always align with FHB severity. Understanding and incorporating information on genetic and phenotypic diversity of FHB isolates used in screening programs should facilitate development of barley varieties that are resistant to FHB under diverse environmental conditions.