The fingerprinting methods referred to as MSP-PCR (microsatellite-primed polymerase chain reaction) and rep-PCR (repetitive-sequence-based polymerase chain reaction) were used to discriminate between species of Botryosphaeriaceae (Fungi, Ascomycota). Several primers were tested with both methods and each primer enabled clear differentiation of all the species tested. Cluster analysis of banding patterns of the isolates corresponded well with known species delineations based on morphology and phylogenetic analysis. The methods described in this paper provide simple and rapid procedures that can be used for routine differentiation of Botryosphaeriaceae isolates at the species level. The two methods are also useful for studying intraspecific variability.