It is difficult to accurately identify Mycosphaerella species associated with leaf diseases of Eucalyptus based on morphological characters, as there is considerable overlap between very similar species and subspecies, and isolation from the host is not easy. Thus, a PCR and RFLP assay based on the ITS region of nr DNA was developed for the rapid detection and differentiation of M. nubilosa, M. cryptica and two non-sporing unidentified Mycosphaerella species isolated from the foliage of trees in resistant and susceptible families of E. globulus in a seed orchard at Kinglake West, Victoria, Australia. The M. nubilosa primer pair MNF/MNR was highly specific. A PCR-RFLP system based on the primer pair MCF/MCR, coupled with two restriction enzymes (DdeI and Tru1I), differentiated M. cryptica, M. nubilosa, M. tasmaniensis and M. aff. vespa. One of the unidentified field-isolated Mycosphaerella species was identified as M. grandis on the basis of ITS sequence data while the other species remains unidentified. A PCR-RFLP system based on the primer pair U1F/U1R, coupled with the restriction enzyme StyI, differentiated between the two unidentified species. Unexpectedly, unlike isolation and culture studies, these assays detected M. nubilosa, M. cryptica and M. grandis in all single lesions examined on both juvenile and adult leaves, and on both highly resistant and highly susceptible E. globulus trees at this site.