Abstract: In the process of tobacco disease investigation in Chongqing, an unknown leaf spot disease on tobacco was found in Runxi Town, Pengshui County, Chongqing. To identify the taxonomy of the pathogen, typical diseased samples were collected from the affected tobacco fields. After tissue isolation, strain purification and pathogenicity determination by Koch's rule, this leaf spot disease was diagnosed and identified with rDNA-ITS molecular identification method. The results showed that the spot of the leaf disease was irregular and surrounded by a faint yellow halo, the center of the diseased part was gray white and densely covered with black spots. The strain psrx-1 isolated from the samples was pathogenic to healthy tobacco plants. The morphological characteristics of the pathogenic strains could be described as follows: the sporodochium was cushion-shaped, dark brown, spherical or hemispherical; the conidial stems were closely arranged, short and upright; the conidia were dark brown, spherical or nearly spherical without diaphragm, and the surface displayed granular bulges or wrinkles. Through rDNA-ITS gene sequence alignment analysis, this disease was identified as tobacco leaf spot disease caused by Epicoccum sp., which is a new tobacco disease reported for the first time in Chongqing tobacco-planting area.