Tamburic-Ilincic, L., Wragg, A. and Schaafsma, A. 2015. Mycotoxin accumulation and Fusarium graminearum chemotype diversity in winter wheat grown in southwestern Ontario. Can. J. Plant Sci. 95: 931-938. Fusarium head blight caused by Fusarium graminearum is a serious disease of wheat (Triticum aestivum L.). Deoxynivalenol (DON) is the mycotoxin most commonly detected in contaminated wheat grain in Ontario. A chemotype shift from 15-acetyldeoxynivalenol (15-ADON) to 3-acetyldeoxynivalenol (3-ADON) has been reported in some parts of North America. The objectives of this study were to investigate the mycotoxins accumulation and Fusarium graminearum chemotype diversity in different winter wheat cultivars grown in southwestern (SW) Ontario. Twenty winter wheat grain samples from SW Ontario were tested for DON concentrations in 2008, 24 in each of 2009 and 2010, 42 in 2011 and 36 in 2013 growing seasons. DON levels in grain ranged from none detected to 33 parts per million (ppm). The highest averaged levels of DON were detected in the 2013 season (19.8 ppm at Centralia, 11.8 ppm at Inwood and 4.1 ppm at Ridgetown). In addition to total DON, H-T2, T-2 and nivalenol mycotoxins were detected in low concentrations in a small number of samples collected in 2009 and/or 2013. The 15-ADON analogs were detected at approximately 1.5% of the total DON in harvested wheat grain in 2013. Ninety-seven percent of F. graminearum isolates, tested in 2010 using the polymerase chain reaction (PCR) method, were 15-ADON chemotype, while just 3% of isolates were the 3-ADON chemotype. Overall, there was no evidence in this study to support a shift from 15-ADON to 3-ADON chemotypes of F. graminearum in SW Ontario in 2010. It is recommended to continue monitoring mycotoxin accumulation and F. graminearum chemotype diversity so any changes can be detected early and addressed.