Two PCR primers (MSU1 and MSU7) were designed to amplify part of the mt SSU rDNA gene from filamentous ascomycetes. The MSU1-MSU7 and MSU1-NMS2 primer pairs were tested with 32 isolates of 23 species in 10 genera. Minimum evolution, parsimony, and likelihood methods were used for the phylogenetic analyses of mt SSU rDNA sequences from Botryosphaeria species and related taxa. These analyses indicated that: Botryosphaeria is not monophyletic; the separation of Botryosphaeria from Guignardia is supported; except for B. vaccinii, other Botryosphaeria species and related taxa form a monophyletic group with strong jackknife support; Diplodia pinea f. sp. cupressi is different from RAPD marker groups A and B of Sphaeropsis sapinea; the anamorph genera Sphaeropsis and Diplodia are not indicative of close relationships among different species; and the name B. stevensii may have been applied to more than one species. Botryosphaeria needs additional study and substantial taxonomic revision.