Abstract: Brown spot disease is one of the primary factors affecting safety of leaf tobacco quality in Changsha tobacco-growing region. To investigate the occurrence and identify the pathogenic species causing tobacco brown spot disease, a comprehensive survey was conducted on incidence rate and disease index of brown spot disease across the tobacco production towns in Changsha. Results showed that the incidence rate and disease index ranged 0.56%-16.67% and 0.06-5.43 respectively. Through combined morphological and molecular biological identification of isolated pathogens, the study revealed that the causative agents were Alternaria alternata and A. tenuissima, with the detection rate of A. tenuissima up to 75.00%. Further in this study, rDNA-ITS gene was confirmed to be excessive conservation for species-level differentiation within the Alternaria genus, whereas the plasma membrane ATPase gene displayed concordance with morphological identification results, proving suitable for interspecies discrimination. In conclusion, the pathogens of tobacco brown spot disease in Changsha tobacco-growing region exhibit high diversity, with Alternaria alternata being predominant.