Fusarium graminearum causes fusarium head blight (FHB) of wheat and gibberella ear rot (GER) of corn in Canada and also contaminates grains with trichothecene mycotoxins. Very little is known about trichothecene diversity and population structure of the fungus from corn in Ontario, central Canada. Trichothecene genotypes of F. graminearum isolated from corn (n = 452) and wheat (n = 110) from 2010 to 2012 were identified. All the isolates were deoxynivalenol (DON) type. About 96% of corn isolates and 98% of wheat isolates were 15-acetyl deoxynivalenol (15ADON) type. The fungal population structures from corn (n = 313) and wheat (n = 73) were compared using 10 variable number tandem repeat (VNTR) markers. The fungal populations and subpopulations categorized based on host, cultivar groups, years and geography showed high gene (H = 0.818–0.928) and genotypic (GD = 0.999–1.00) diversity. Gene flow was also high between corn and wheat population pairs (Nm = 8.212), and subpopulation pairs within corn (Nm = 7.13–23.614) or wheat (Nm = 19.483) populations. Phylogenetic analysis revealed that isolates from both hosts were F. graminearum clade 7. These findings provide baseline data on 3-acetyl deoxynivalenol (3ADON) and 15ADON profiles of F. graminearum isolates from corn in Canada and are useful in evaluating mycotoxin contamination risks in corn and wheat grains. Understanding the fungal genetic structure will assist evaluation and development of resistant cultivars/germplasm for FHB on wheat and GER on corn.