Twenty-four Australian strains of infectious bursal disease virus (IBDV) were characterized by reverse transcription/polymerase chain reaction-restriction fragment length polymorphism and compared with previously published overseas strains. A primer pair designed to amplify a 743 base pair fragment of the VP2 gene was used and restriction fragment length polymorphism profiles were determined for each strain using three restriction enzymes, Bst NI, MboI and SspI. Australian strains comprised 12 molecular groups that were unique and distinct from overseas IBDV strains. A specific SspI site that is used to predict a very virulent IBDV phenotype was absent from all Australian strains, contrary to a previous finding by Jackwood and Sommer (1999). One Australian strain (N1/99) contained an SspI site; however, this was located at a different position than that found in very virulent IBDV strains. The results demonstrate that restriction fragment length polymorphism can be used to rapidly differentiate Australian IBDV strains from overseas strains. However, the existence of a large number of molecular groups might preclude its effectiveness for inter-strain differentiation.