The filamentous fungal genus Stemphylium (Anamophic Pleospora) is often found on various crops, and especially the common animal feed plant Medicago sativa (alfalfa) is often infected by this plant pathogen. With this in mind it is important to consider what consequences such a contamination can have, e.g. production of mycotoxins. (Firsvad et al. (2009)) A clade of Stemphylium spp. i.e. S. herbarum, S. alfalfae, S. sedicola, S. tomatonis and S. vesicariumare troublesome to distinguish as they share both morphological and molecular characteristics. This study has focused on using chemotaxonomy via HPLC-UV/VIS-MS analysis as a third method for identification and to investigate the chemical potential of the genus Stemphylium. A total of 253 unique compounds were used for chemotaxonomy and the majority of these were unknown compounds.