Fusarium graminearum is the most important pathogen of maize and winter wheat in Canada, resulting in yield loss and mycotoxin contamination. Fungicides are essential to manage F. graminearum in cereal crops. Until pydiflumetofen became commercial, the only available fungicides were triazoles, limiting the potential to manage fungicide resistance. Fusarium graminearum isolates were surveyed from wheat, grain maize and overwintering maize stalks in southern Ontario. Chemotype and the baseline sensitivity to pydiflumetofen were determined in addition to sensitivity to common fungicides used to control F. graminearum. Fusarium graminearum has several strains producing different trichothecenes called chemotypes. In Ontario, 73.3% of the F. graminearum isolates were 3ANX chemotypes producing 7α-hydroxy, 15-deacetylcalonectrin, in addition to 15-acetyldeoxynivalenol and deoxynivalenol; the remaining isolates were 15ADON chemotypes producing only 15-acetyldeoxynivalenol and deoxynivalenol. Chemotype was not affected by isolate source (wheat grain, maize grain, maize stalks) nor by location. All isolates were sensitive to pydiflumetofen and fungicide products containing pydiflumetofen. The mean EC50 for isolates exposed to pydiflumetofen was 0.05 µg mL−1 (range 0.01–0.10 µg mL−1). The mean EC50 for isolates exposed to Miravis®Ace (containing pydiflumetofen and propiconazole), Miravis®Neo 300SE (containing pydiflumetofen, azoxystrobin and propiconazole), Caramba® (containing metconazole), Proline® (containing prothioconazole) and Prosaro (containing prothioconazole and tebuconazole) was 0.06, 0.13, 0.18, 0.05, 3.75 and 0.30 µg mL−1, respectively. These data will be useful in monitoring the development of fungicide resistance.