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Fusarium head blight (FHB) is a destructive disease of wheat (Triticum aestivum L). FHB reduces yield and grain quality and causes accumulation of the mycotoxin deoxynivalenol (DON) in grain. Cultivar resistance is one of the most effective management strategies for FHB. Experiments were conducted to 1) identify winter wheat cultivars with resistance to FHB and DON accumulation, 2) determine the effect of winter wheat cultivar on the relationship between FHB and DON concentration, and 3) identify the major species of Fusarium causing FHB in Nebraska and characterize its isolates. Differences (P ≤ 0.05) were detected among cultivars in FHB index, Fusarium-damaged kernels, DON, and yield. The cultivars Alliance, Harry, Hondo, Infinity CL, and Overland were moderately resistant to FHB and DON accumulation. Harry was resistant to FHB but susceptible to DON accumulation. Overley, Jagalene, Wesley, and 2137 were susceptible to FHB and DON accumulation. The relationship between FHB severity and DON concentration was linear and positive regardless of cultivar. However, regression slopes indicated that this relationship was cultivar dependent. Forty of 41 isolates of Fusarium obtained from infected winter wheat kernels in grain collected from elevators and fields in Nebraska were identified by polymerase chain reaction (PCR) morphological characteristics as Fusarium graminearum Schwabe (Teleomorph: Gibberella zeae (Schwein.) Petch). Seventeen selected isolates differed (P ≤ 0.05) in the number and size of perithecial units (single perithecium or clusters of perithecia) produced in culture and seven selected isolates differed (P ≤ 0.05) in aggressiveness on wheat spikes and detached leaves. Based on aggressiveness on wheat spikes, the seven isolates were grouped into three categories: 1) highly aggressive (isolates 103, 110, and 119), 2) moderately aggressive (isolates 91 and 98), and 3) weakly aggressive (isolates 90 and 97).