Abstract: Aiming to clarify the species and biological characteristics of pathogenic fungal leaf spot diseases in Zhongshan County, Hezhou City, Guangxi Zhuang Autonomous Region, the pathogenic fungi were isolated by tissue isolation and its pathogenicity was determined by inoculation of living wounds. The molecular identification of pathogenic fungi was performed by PCR amplification of their internal transcribed spacer region of the ribosomes (ITS), actin (ACT), and chitin synthase (CHS). The pathogen species were identified by combining morphological characteristics, and mycelial growth rate method was employed to determine the biological characteristics of pathogens. Results of cluster analysis showed that the pathogen and Collettrichum truncatum were clustered into one team, and the support rate reached 100%. Combined with morphological characteristics, the pathogen was identified as C. truncatum, which is the first report of tobacco anthracnose caused by C. truncatum parasitising tobacco. The determination of pathogen’s biological characteristics showed that the pathogen exhibited strong adaptability to environment. The optimal medium, temperature, light condition, carbon source, nitrogen source was nutrient agar (NA), 30 ℃, total darkness, starch, peptone, respectively. The most rapid growth of pathogen was observed at pH 7, and the lethal temperature of pathogen was 53 ℃ for 10 min. The results of the study present the diversity of pathogens causing tobacco anthracnose, and provide a theoretical basis for the timely accurate control and further research of the disease.