From 2022 to 2023 crop year, a stalk disease resembling tobacco canker was observed on tobacco plants in Yongzhou County of Hunan Province. In order to identify the pathogen and clarify the biological characteristics of the disease, a tissue separation method was used to isolate and purify the pathogen from diseased stalks, then morphological observation and verification of Koch's postulates were carried out. The biological characteristics of the pathogen were investigated. Morphological and phylogenetic analyses of the rDNA-ITS sequence confirmed the pathogen asBotryosphaeria dothidea. Investigation into the biological characteristics ofBotryosphaeria dothidearevealed an optimal mycelial growth temperature of 30 ℃, pH of 7, with glucose and bovine extract identified as the preferred carbon and nitrogen sources, respectively. This study represents the first report of tobacco canker caused byBotryosphaeria dothideain Hunan Province.