Abstract: To clarify the pathogen causing leaf spots of tobacco in Guizhou Province and its biological characteristics, the pathogen was obtained by using the method of tissue isolation. Its pathogenicity was tested in vitro with inoculated leaves, and the pathogen was classified and identified with morphological characteristics combined with molecular biological methods. The biological characteristics of the pathogen were also studied. The results showed that the pathogen clustered with Curvularia clavata in the phylogenetic tree based on ITS sequence, and the support rate was 100%. Combined with the morphological characteristics, the pathogen was identified as Curvularia clavata. The study of biological characteristics showed that the optimum temperature for mycelium growth was 28℃, the optimum pH was 8, the fastest growth was on oat agar (OA), and it was most favorable for mycelium growth when soluble starch was used as carbon source and beef powder as nitrogen source, and the lethal temperature of mycelium was 53℃ and 10 min. The light had no significant effect on mycelial growth of Curvularia clavata. The results can provide a theoretical basis for the control of tobacco Curvularia leaf spot.