Abstract: To investigate pathogen species causing tobacco black root rot and their pathogenicities in Henan Province, tobacco plants with typical black root rot and their rhizosphere soil were sampled to isolate pathogens by organization separation method and carrot disc culture. The species of pathogens were identified by observing morphology characteristics and analyzing sequences of rDNA-ITS, their pathogenicities were detected by irrigating the roots of tobacco seedlings. The results showed that four strains, YYTB, LNTB, JXTB and JYTB, were obtained by carrot disc culture and two white mutant strains of JXTB' and LNTB' were obtained during purifying JXTB and JYTB on PDA medium. The morphological characteristics of YYTB, JYTB, JXTB and LNTB were basically consistent with the colonies with gray to olive-green in color, endogenous conidiophores were colorless to brown in color, solitary or clustered; endogenous conidium was colorless and rod-shaped single spore with two round ends; chlamydospores were dark brown, clustered or solitary. The morphological characteristics of JYTB' and LNTB' were basically consistent, their colonies were white in color, and chlamydospores were colorless; their conidiophores and conidium featured a similar morphology to those of YYTB, JYTB, JXTB and LNTB. The homology of rDNA-ITS sequences was above 99% between six isolate strains and Thielaviopsis basicola (GenBank accession numbers:AF275482 and U97334). The six isolate strains were pathogenic to tobacco seedlings of cv. Yuyan 6, however the pathogenicities of JXTB' and LNTB' were weaker. Therefore, the pathogen of tobacco black root rot was identified as Thielaviopsis basicola (Berk. et Br.) Ferraris.