The majority of Eucalyptus species are native to Australia, but worldwide there are over 3 million ha of exotic plantations, especially in the tropics and subtropics. Of the numerous known leaf diseases, three species of Phaeophleospora can cause severe defoliation of young Eucalyptus; Phaeophleospora destructans, Phaeophleospora eucalypti and Phaeophleospora epicoccoides. Phaeophleospora destructans has a major impact on seedling survival in Asia and has not, as yet, been found in Australia where it is considered a serious threat to the biosecurity of native eucalypts. It can be difficult to distinguish Phaeophleospora species based on symptoms and micromorphology and an unequivocal diagnostic tool for quarantine purposes would be useful. In this study, a multiple gene genealogy of these Phaeophleospora species and designed specific primers has been constructed to detect their presence from leaf samples. The phylogenetic position of these Phaeophleospora species within Mycosphaerella was established. They are closely related to each other and to other important Eucalyptus pathogens, Mycosphaerella nubilosa, Mycosphaerella cryptica and Colletogloeopsis zuluensis. The specific primers developed can now be used for diagnostic and screening purposes within Australia.