Ray blight, a destructive disease of Asteraceae worldwide, is caused by three morphologically similar but phylogenetically distinct species; Stagonosporopsis chrysanthemi, S. inoxydabilis and S. tanaceti. Stagonosporopsis chrysanthemi has been reported as a specific pathogen of chrysanthemum while S. inoxydabilis has been found associated with various Asteraceae. Stagonosporopsis tanaceti has only been reported in Australia, causing substantial crop loss on pyrethrum. All three species were shown to infect and cause disease on in vitro grown pyrethrum plants, hence, S. chrysanthemi and S. inoxydabilis may pose a significant biosecurity threat to the Australian pyrethrum industry. All these Stagonosporopsis species are also Level 2 quarantine pathogens in Europe. Rapid and accurate detection and differentiation of these species is a priority for ray blight management in Australia and in Europe. Accordingly, three species-specific PCR-based assays, targeted to the intergenic spacer of the nuclear ribosomal DNA, were developed. The specificity of each assay was confirmed against 21 Stagonosporopsis spp. as well as 14 pathogenic and saprophytic fungal species commonly found in association with pyrethrum in Australia. The primers were highly sensitive and specific to the target species, detecting down to 4 fg of genomic DNA. These primers were further used in a multiplex PCR to differentiate the presence of the three Stagonosporopsis spp. based on variable sized amplicons in a single reaction.