A PCR-based technique based on the ITS1-5.8s-ITS2 domain of the rRNA gene for identifying five species associated with Mycosphaerella leaf disease (MLD) of eucalypts was developed. Primer pairs MC2F and MC2R; ML1F and ML1R; MM1F and MM1R; MN1F and MN1R; and MP1F and MP1R amplified a product for DNA extracted from their single target species, those being M. cryptica, M. lateralis, M. marksii, M. nubilosa and M. parva, respectively. The possibility of false positive amplification by each primer pair was tested in reactions with DNA extracts from 16 other Mycosphaerella species associated with eucalypts and against non-infected Eucalyptus globulus leaves. Under the PCR conditions used, there were no false positive amplifications of the 16 non-target Mycosphaerella species, or from non-symptomatic E. globulus leaves for the primer pairs ML1F and ML1R; MM1F and MM1R; MN1F and MN1R; and MP1F and MP1R. The primer pair MC2F and MC2R amplified a 402 nt product from both the target M. cryptica and non-target M. nubilosa. However, these two species were differentiated by digesting the product with the restriction enzyme Sacc II which resulted in a single 402 nt product for M. cryptica, and two products of 78 and 324 nt for M. nubilosa. All of the primers were able to detect their target Mycosphaerella species from Eucalyptus globulus lesions. PCR reactions with these primers on DNA extracted from Mycosphaerella lesions confirmed the presence of all five species from leaf material collected from three plantations in Western Australia.