Species of Pestalotiopsis occur commonly as plant pathogens, and represent a fungal group known to produce a wide range of chemically novel, diverse
metabolites. In the present study, we investigated 91 Pestalotiopsis isolates from the CBS-KNAW Fungal Biodiversity Centre (CBS) culture collection. The phylogeny of
the Amphisphaeriaceae was constructed based on analysis of 28S nrRNA gene (LSU) sequence data, and taxonomic changes are proposed to reflect more natural
groupings. We combined morphological and DNA data, and segregated two novel genera from Pestalotiopsis, namely Neopestalotiopsis and Pseudopestalotiopsis. The
three genera are easily distinguishable on the basis of their conidiogenous cells and colour of their median conidial cells. We coupled morphological and combined
sequence data of internal transcribed spacer (ITS), partial β-tubulin (TUB) and partial translation elongation factor 1-alpha (TEF) gene regions, which revealed 30 clades
in Neopestalotiopsis and 43 clades in Pestalotiopsis. Based on these data, 11 new species are introduced in Neopestalotiopsis, 24 in Pestalotiopsis, and two in
Pseudopestalotiopsis. Several new combinations are proposed to emend monophyly of Neopestalotiopsis, Pestalotiopsis and Pseudopestalotiopsis.