The tomato inbred line YNAU335, previously behaved immune to tomato spotted wilf virus (TSWV), exhibited with TSWV symptoms which indicated that the resistance of YNAU335 to TSWV had been overcome. To clarify the resistancebreaking determinant, we firstly identified both the infected plants of susceptible 96172I and resistancebreaking YNAU335 inbred lines with RTPCR method to clone the nucleocapsid protein (NP) and movement protein (MP) genes, and the target genes of positive clones were further analyzed by sequencing. The results were shown as follows. (1) The NP and MP genes of TSWV existed in the two inbred lines, and five NP (Accession No. MK628735MK628739) and three MP (Accession No. MK883723, MK883724 and MK887284) polymorphic gene sequences were also gained. (2) All the gene sequences mentioned were cloned from 96172I infected material, whereas, YNAU335 infected material possessed the three NP and one MP of the above gene sequences. (3) Amino acid mutations in the NP and MP of YNAU335 inbred line were more analyzed. Four specific mutations might be related to the resistancebreaking event of YNAU335 inbred line: substitution G to V at position 18(G18V), T36I and L39R in the NP, and E274K in the MP. (4) Phylogenetic analysis revealed that the five NP and three MP, and the registered NP and MP from Yunnan province were clustered in different branches, which indicated the presence of abundant genetic diversity of TSWV in Yunnan province.