Abstract: In 2020 and 2021, a new type of tobacco leaf disease broke out in the main tobacco producing areas of Hubei Province. The pathogen was analyzed through isolation and morphological observation, microscopic examination of hyphae and verification of Koch's postulates, extraction of the DNA of 30 strains and amplification of the rDNA- ITS sequence by PCR with universal primers ITS1/ITS4. The MEGA-X software was used for gene sequence alignment and phylogenetic tree construction, and the PCR specificity of rDNA-ITS genes of 30 strains was identified with Rs1F2/Rs2R1 as primers. The results showed that the pathogen was identified as Rhizoctonia solani Kühn, all 30 strains belonged to the R. solani AG-3 anastomosis group and with a 504 bp fragment unique to AG-3 anastomosis group. Therefore, the disease was identified as tobacco target spot. This is the first time that the pathogen was isolated from tobacco producing areas in Hubei Province.