Abstract: To investigate the pathogen species causing tobacco black root rot in Anhui, tobacco plants infected with typical black root rot were sampled to isolate pathogens via carrot disc culture. The species of pathogens were identified by morphological observation and rDNA-ITS sequencing, their pathogenicities were detected by irrigating the roots of tobacco seedlings, and the biological characteristics of pathogens were researched. The results showed that the pathogen of tobacco black root rot was identified as Thielaviopsis basicola (Berk. et Br.) Ferraris and it was pathogenic to cv. Yunyan 87. The results of biological characteristics indicated that the optimal medium, pH, temperature for mycelial growth and spore germination of T. basicola were V8, 6 and 25℃, respectively. The lethal temperature of conidiospore was 53℃. The pathogen could effectively utilize carbon sources (starch, maltose, glucose, etc.) and nitrogen sources (carbamide, zinc nitrate, etc.).