To understand the organization of the mating type locus of Stagonosporopsis tanaceti and Stagonosporopsis chrysanthemi, and its potential role in the epidemiology of ray blight of pyrethrum and chrysanthemum, respectively, the mating type (MAT) locus of these species was cloned and characterized using PCR-based techniques. The complete MAT locus of each species was cloned and annotated including complete and/or partial hypothetical genes flanking the idiomorphs. Analysis of the MAT locus organization indicated that S. chrysanthemi is likely homothallic with both MAT1-2-1 and MAT1-1-1 co-located within the idiomorph, and this was supported by production of the teleomorph in cultures of single-conidial-derived isolates. Sequencing of the MAT locus and flanking genes of S. tanaceti demonstrated that only a single MAT gene, MAT1-1-1, was located within this idiomorph and suggesting that S. tanaceti is heterothallic. MAT-specific PCR primers were developed and used to determine mating type of isolates sampled from diseased pyrethrum fields in Australia. These results indicated that only one mating type of S. tanaceti was present in Tasmania, Australia. The absence of a second mating type suggests that this species does not reproduce sexually in Tasmania, Australia and that ascospores are unlikely to be a source of inoculum for ray blight of pyrethrum. The MAT-specific PCR assay will be a valuable tool to distinguish mating types present among isolates of S. tanaceti, to monitor populations of S. tanaceti for the introduction of a second mating type and to differentiate S. tanaceti from S. chrysanthemi.