Inter simple or short sequence repeat (ISSR) fingerprinting was used to differentiate Botryosphaeria species and related anamorphic fungi. Fifty-one isolates representing 10 different Botryosphaeria species and related taxa, including two RAPD marker groups and one special form of Sphaeropsis sapinea were investigated using five ISSR primers. A total of 171 fingerprint fragments were generated from these isolates, and sizes of fragments were mostly between 200 bp and 1650 bp. Both cluster and principal coordinate analyses were used to examine separately the fingerprint data from the two previously proposed sections Hyala and Brunnea of Botryosphaeria. The results of each analysis indicated that B. dothidea and B. ribis are distinct; B. ribis and B. parva are closely related, but separable; B. mamane is distinct from other Botryosphaeria species with similar morphology or ITS sequences; the A and B RAPD marker groups of S. sapinea and S. sapinea f. sp. cupressi are probably three distinct species; B. obtusa and S. sapinea B group are closely related but distinct; and the name B. stevensii may have been applied to more than one species. This study has shown that ISSR fingerprinting can be a powerful tool for differentiating closely related filamentous fungi with very similar morphologies or ITS sequences, dissecting species complexes, and justifying newly described species.