Members of Botryosphaeria encompass important plant pathogens, saprobes and endophytes on a wide range of woody hosts worldwide. Botryosphaeria species are difficult to differentiate due to the overlapping morphological characteristics and the molecular data analyses are necessary recently when species identification is carried out. In this study, 28 Botryosphaeria isolates were obtained from decaying woody hosts in six nature reserves in Guizhou province, China. Based on both morphological characteristics and molecular analysis of combined ITS and tef1-α sequence data, four known species (Botryosphaeria dothidea, B. minutispermatia, B. sinensia and B. wangensis) are identified and one new species B. guttulata is introduced. Botryosphaeria sinensia (32% of the isolates obtained from various hosts) is the abundant species, followed by B. dothidea (28.5% of the isolates), B. guttulata (28.5% of the isolates), B. minutispermatia (7% of the isolates) and B. wangensis (4% of the isolates). These results represent the first study of Botryosphaeria species associated with woody hosts from nature reserves in Guizhou province, China. Our findings indicate that there is a potential of Botryosphaeria species remain to be discovered in this unique landform (Karst formations) in Guizhou province, China.