The Botryosphaeriaceae is a species-rich family that includes pathogens of a wide variety of plants, including species of Eucalyptus. Recently, during disease surveys in China, diseased samples associated with species of Botryosphaeriaceae were collected from plantation Eucalyptus and other plants, including Cunninghamina lanceolata, Dimocarpus longan, Melastoma sanguineum and Phoenix hanceana, which were growing adjacent to Eucalyptus. In addition, few samples from Araucaria cunninghamii and Cedrus deodara in two gardens were also included in this study. Disease symptoms observed mainly included stem canker, shoot and twig blight. In this study, 105 isolates of Botryosphaeriaceae were collected from six provinces, of which 81 isolates were from Eucalyptus trees. These isolates were identified based on comparisons of the DNA sequences of the internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS), and partial translation elongation factor 1-alpha (tef1), β-tubulin (tub), DNA-directed RNA polymerase II subunit (rpb2) and calmodulin (cmdA) genes, the nuclear ribosomal large subunit (LSU) and the nuclear ribosomal small subunit (SSU), and combined with their morphological characteristics. Results showed that these isolates represent 12 species of Botryosphaeriaceae, including Botryosphaeria fusispora, Cophinforma atrovirens, Lasiodiplodia brasiliense, L. pseudotheobromae, L. theobromae and Neofusicoccum parvum, and six previously undescribed species of Botryosphaeria and Neofusicoccum, namely B. pseudoramosa sp. nov., B. qingyuanensis sp. nov., B. wangensis sp. nov., N. hongkongense sp. nov., N. microconidium sp. nov. and N. sinoeucalypti sp. nov. Aside from B. wangensis, C. atrovirens and N. hongkongense, the other nine Botryosphaeriaceae species were isolated from Eucalyptus trees in South China. Botryosphaeria fusispora (26 % of the isolates from Eucalyptus) is the dominant species, followed by L. pseudotheobromae (23 % of the isolates from Eucalyptus). In addition to species found on Eucalyptus trees, we also found B. pseudoramosa on M. sanguineum; B. wangensis on C. deodara; C. atrovirens on D. longan; L. theobromae on C. lanceolata, D. longan and P. hanceana; and N. hongkongense on A. cunninghamii. Pathogenicity tests showed that the 12 species of Botryosphaeriaceae are pathogenic to three Eucalyptus clones and that Lasiodiplodia species are the most aggressive. The results of our study suggest that many more species of the Botryosphaeriaceae remain to be discovered in China. This study also provides confirmation for the wide host range of Botryosphaeriaceae species on different plants.