Positive-negative PCR assays based on the genes involved in the trichothecene biosynthesis pathway are useful in assessing the risk of trichothecene contamination in grain and are important in epidemiological studies. A single PCR detection method based on the structural gene sequence of TRI13 gene has been developed to predict the 3-ADON, 15-ADON and NIV chemotypes in China. The chemotypic differences are based on the deletions within the TRI13 gene. The objective of this study was to assess the reliability of using this single primer based on the TRI13 gene to differentiate the F. graminearum chemotypes in Canada. In this study, we found that, this single PCR detection method based on the deletions in the TRI13 gene cannot be used to differentiate the 3-ADON and 15-ADON chemotypes in the Canadian F. graminearum isolates; further sequence analysis of the PCR products confirmed that both Canadian 3-ADON and 15-ADON chemotypes have the 61 bp deletion in the TRI13 gene. This 61 bp deletion was absent in the Chinese 3-ADON isolates. Therefore these findings revealed that there are genetic differences between the examined 3-ADON F. graminearum isolates from Canada and China. The observed genetic differences between the 3-ADON chemotype populations in Canada and China may be resulted from a random mutation (insertion/deletion) that took place in one of the populations and accumulated due to genetic drift and/or selection.