Abstract: In order to clarify the types of pathogens causing tobacco brown spot disease in Jilin and Heilongjiang Provinces, this study isolated and identified pathogens of tobacco brown spot disease from 15 major tobacco producing areas in Jilin Province and 7 major tobacco producing areas in Heilongjiang Province. A total of 312 pathogenic strains were isolated and identified. Four species of Alternaria were identified through morphological and molecular biological identification, including 230 strains of Alternaria alternata, 78 strains of Alternaria tenuissima, 2 strains of Alternaria yaliinficiens, and 2 strains of Alternaria longipes. A. alternata, A. tenuissima and A. yaliinficiens were identified in Jilin Province and A. longipes, A. alternata and A. tenuissima were identified in Heilongjiang Province. A. alternate and A. tenuissima were both identified in the two Province and A. alternata was the dominant species in both Province. In addition, five gene sequence fragments (ITS, GDP, ACT, EF, RPB2) were used for molecular biological identification in this study, in which the GDP gene can only identify A. alternata and A. tenuissima, RPB2 gene can identify all four species of Alternaria, while ITS, EF, ACT gene sequences are more conservative. So ITS, EF and ACT gene sequences are not suitable for distinguishing the various species of Alternaria. Through this study, the types of pathogens causing tobacco brown spot disease in Jilin Province and Heilongjiang Province were identified, which laid the foundation for the next stage of targeted prevention and control.