| Summary: | <i>Alternaria</i> spp. occur as plant pathogens worldwide under field and storage conditions. They lead to food spoilage and also produce several classes of secondary metabolites that contaminate the food production chain. From a food safety perspective, the major challenge of assessing the risk of <i>Alternaria</i> contamination is the lack of a clear consensus on their species-level taxonomy. Furthermore, there are currently no reliable DNA sequencing methods to allow for differentiation of the toxigenic potential of these fungi. Our objective was to determine which species of <i>Alternaria</i> exist in Canada, and to describe the compounds they make. To address these issues, we performed metabolomic profiling using liquid chromatography high-resolution mass spectrometry (LC-HRMS) on 128 Canadian strains of <i>Alternaria</i> to determine their chemotaxonomy. The <i>Alternaria</i> strains were analyzed using principal component analysis (PCA) and unbiased <i>k</i>-means clustering to identify metabolites with significant differences (<i>p</i> < 0.001) between groups. Four populations or ‘chemotypes’ were identified within the strains studied, and several known secondary metabolites of <i>Alternaria</i> were identified as distinguishing metabolites, including tenuazonic acid, phomapyrones, and altenuene. Though species-level identifications could not be concluded for all groups through metabolomics alone, <i>A. infectoria</i> was able to be identified as a distinct population.
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