Abstract: To understand the diversity of mycovirus in the pathogen causing tobacco target spot, dsRNA screening and high-throughput sequencing were performed on the pathogen, and homology searches and phylogenetic analyses were conducted on the obtained candidate viral sequences to determine the viral species and taxonomic status, the analyses of growth rate and pathogenicity were carried out on the virus-carrying strains. The results show that among 148 strains of the tobacco target spot, 14 strains were found to carry dsRNA, which were identified as 28 distinct mycoviruses via sequencing. They belonged to five viral families: Barnaviridae, Benyviridae, Mitoviridae, Narnaviridae, and Partitiviridae. Comparison of growth rate and pathogenicity revealed that no significant difference occurred in growth rate among the virus-carrying strains, yet their virulence exhibited differentiation. The effects of different viruses and their combinations on the function of host strains remains to be further explored.