In this study, fungal specimens of the family Diatrypaceae were collected from karst areas in Guizhou, Hainan and Yunnan Provinces, China. Morpho-molecular analyses confirmed that these new collections comprise a new genus Pseudodiatrype, three new species (Diatrype lancangensis, Diatrypella pseudooregonensis and Eutypa cerasi), a new combination (Diatrypella oregonensis), two new records (Allodiatrype thailandica and Diatrypella vulgaris) from China and two other known species (Neoeutypella baoshanensis and Paraeutypella citricola). The new taxa are introduced, based on multi-gene phylogenetic analyses (ITS, β-tubulin), as well as morphological analyses. The new genus Pseudodiatrype is characterised by its wart-like stromata with 5–20 ascomata immersed in one stroma and the endostroma composed of thin black outer and inner layers of large white cells with thin, powdery, yellowish cells. These characteristics separate this genus from two similar genera Allodiatrype and Diatrype. Based on morphological as well as phylogenetic analyses, Diatrype lancangensis is introduced as a new species of Diatrype. The stromata of Diatrype lancangensis are similar to those of D. subundulata and D. undulate, but the ascospores are larger. Based on phylogenetic analyses, Diatrype oregonensis is transferred to the genus Diatrypella as Diatrypella oregonensis while Diatrypella pseudooregonensis is introduced as a new species of Diatrypella with 8 spores in an ascus. In addition, multi-gene phylogenetic analyses show that Eutypa cerasi is closely related to E. lata, but the ascomata and asci of Eutypa cerasi are smaller. The polyphyletic nature of some genera of Diatrypaceae has led to confusion in the classification of the family, thus we discuss whether the number of ascospores per asci can still be used as a basis for classification.