Fusarium graminearum is a fungal pathogen of grains resulting in yield loss and more importantly contamination with the mycotoxin deoxynivalenol (DON). In recent years, genetic changes in the F. graminearum population have occured and concerns include the risk of more DON in grain with less visible disease, new toxins and an increase in pathogen fitness and virulence. The aim of this study was to isolate F. graminearum from wheat and corn collected during the 2015-2017 Ontario harvests, and compare genotype and chemotype analysis. Representative strains were examined using PCR assays to determine genotype, and chemical analysis to determine chemotype. All strains were determined to be 15ADON through genotype analysis. However, chemical analysis showed co-production of 15ADON and the new trichothecene 3ANX for more than 50% of the strains. These findings demonstrate the existing probes to assess genotype cannot reliably predict chemotype of the population of F. graminearum in Ontario.