This study aimed to assess whether pathogenic Fusarium graminearum isolates from wheat and maize were more aggressive on their host of origin and whether aggressiveness was influenced further by B-trichothecene chemotype. Fifteen isolates were selected from a contemporary collection of isolates surveyed in New York in 2011 to 2012 to represent diversity of host of origin and chemotype. Three pathogenicity assays were used to evaluate and compare these isolates. Fusarium head blight (FHB) severity and trichothecene production in wheat, and maize seedling blight were evaluated in greenhouse inoculation experiments, and Gibberella ear rot (GER) severity and trichothecene production were evaluated in maize ears inoculated in the field. Our results showed among F. graminearum isolates a wide variation in aggressiveness and mycotoxin production toward wheat and maize and these isolates could not be structured by their host of origin or by chemotype. Moreover, aggressiveness rank order changed according to the host/organ evaluated. This indicates that relative susceptibility at the seedling stage may not predict susceptibility of ears. Significant correlations were observed of total trichothecenes (deoxynivalenol [DON] and its acetylated derivatives) produced with FHB and GER severity on wheat and maize, respectively. One isolate did not produce DON or ADON in wheat or maize kernels, yet was aggressive on both hosts. Nine of the fifteen isolates produced small amounts of zearalenone (ZON) in maize kernels, but not in wheat kernels, and ZON level was not correlated with GER severity. F. graminearum isolates from New York showed wide variation in aggressiveness and mycotoxin production toward susceptible wheat and maize. Neither host of origin nor trichothecene chemotype appeared to structure the populations we sampled.