Abstract: To reveal the diversity of mycoviruses in Stagonosporopsis cucurbitacearum strains and investigate their biological characteristics, metaviromic analysis and biological characterization were performed on 12 S. cucurbitacearum strains isolated from tobacco leaf spot samples collected in Sanming City, Fujian Province. The results showed that: 1) Metaviromic analysis identified 13 viral contigs which were determined to belong to 12 viruses (2 dsRNA viruses and 10 +ssRNA viruses) by comparative analysis. Among these contigs, 7 exhibited low homology with known viruses, suggesting that they may represent potential novel viruses. 2) Phylogenetic analysis classified these 12 viruses into 7 families, including Mitoviridae, Botourmiaviridae, Partitiviridae, Narnaviridae, Deltaflexiviridae, Fusariviridae and Totiviridae. 3) Biological characterization revealed that 2 strains (M-2 and M-8) exhibited significantly reduced growth rates, abnormal colony morphology, decreased pigment production and markedly attenuated pathogenicity. The findings enriches the types of mycoviruses infecting S. cucurbitacearum, and 2 hypovirulent strains of S. cucurbitacearum with hypovirulence were identified, providing materials and data for the identification and application of hypovirulence-associated mycoviruses in S. cucurbitacearum for tobacco disease control.