Wollemia nobilis is an ancient coniferous tree species that was recently discovered in eastern Australia. This tree is highly threatened due to its limited distribution. No genetic variation has been detected within the wild populations of ~100 adult plants. A recent study has revealed that a species of Botryosphaeria is highly pathogenic to W. nobilis. The aim of the present study was to identify this fungus, as well as Botryosphaeria isolates of unknown identity from other Southern Hemisphere coniferous hosts, Araucaria from New Zealand and Widdringtonia from South Africa. To facilitate their identification, sequence data for the ITS rDNA, as well as the β-tubulin and translation elongation factor 1-α genes were combined to determine the phylogenetic relationship of these isolates with those of known Botryosphaeria spp. Isolates from W. nobilis included two Botryosphaeria spp. The first is closely related to B. ribis, but also shares some unique sequence polymorphisms with B. parva. One isolate grouped with B. australis, but also varied slightly from this taxon in the gene regions analysed. Additional isolates will be needed to determine whether these sequence variations represent speciation events or merely variation within populations of B. ribis and B. australis. In addition to this, B. parva was identified from Araucaria in New Zealand, and B. australis was found on Widdringtonia trees in South Africa. All three reports of these fungi are new records for their various hosts and could represent important pathogens of these trees.