Abstract: To clarify the classification status of pathogenic fungi and the occurrence of tobacco target spot disease in different regions of Chongqing tobacco area, the occurrence of tobacco target spot disease was investigated in Shuishi Town of Qianjiang District, Wulingshan Town of Fuling District, Jielong Town of Wulong District, Sunjia Town of Wanzhou District, Youyang Tujia and Miao Autonomous County and Pengshui Miao and Tujia Autonomous County in Chongqing. The pathogens from typical diseased leaves were isolated and purified by tissue separation method. The morphological characteristics of the purified strains on PDA medium were observed. The pathogenicities of the purified strains were determined by mycelial block inoculation method. Mycelial fusion with the standard strains was observed by slide confrontation method. The universal primers ITS1/ITS4 were used to amplify the rDNA-ITS sequences of 13 purified strains, and the amplified sequences were analyzed and compared using MEGA 11 software to construct a phylogenetic tree. The results showed that the morphological characteristics of the purified strains were consistent with those of Rhizoctonia solani Kühn, and it had pathogenicity to tobacco seedling leaves. The purified strain was found to be completely fused with the mycelium of Rhizoctonia solani AG-3. Blast alignment and phylogenetic tree analysis of the rDNA-ITS sequence showed that the purified strains were clustered on the same branch with Rhizoctonia solani AG-3 with 97% support rate. Therefore, it was determined that the pathogen of tobacco target spot disease in Chongqing tobacco area was Rhizoctonia solani AG-3.