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Fusarium head blight (FHB) is a devastating disease of wheat (Triticum aestivum L.) and other small grain cereals. The disease is caused by several species of Fusarium. This study was conducted to identify the major species of Fusarium causing FHB in Nebraska and to characterize isolates of the species for perithecia and deoxynivalenol (DON) production and for aggressiveness (quantified as disease severity and area under the disease progress curve) on spikes of two winter wheat cultivars. Isolates of Fusarium spp. obtained from wheat spikes and grain collected from FHB-affected winter wheat fields and grain elevators, respectively, in 2007 and 2008 were identified as Fusarium graminearum. The isolates varied widely in perithecia and DON production in vitro and in aggressiveness on wheat spikes of soft red winter wheat cultivars Coker 9835 (FHB susceptible) and VA04W-433 (moderately resistant). Fusarium head blight severity for the three most aggressive F. graminearum isolates was higher in Coker 9835 than in VA04W-433 during the first 7 d after inoculation (DAI) but was comparable in both cultivars from 10 to 21 DAI. Deoxynivalenol concentration and aggressiveness were positively and linearly related (R² ≥ 0.60, P < 0.05). Isolates that produced higher concentrations of DON were more aggressive than those that produced lower concentrations of the toxin regardless of wheat cultivar. [ABSTRACT FROM AUTHOR]