Virus-induced gene silencing (VIGS) is a method for transiently silencing genes in dicot and monocot plants. To evaluate the effects of chilling injury on activity of the MYB80 gene in Solanum lycopersicum, an investigation was conducted by VIGS using TRV-GFP (a modified TRV vector) to silence the target gene. During the chilling treatment (4/12 °C, 8/16-h dark/light photoperiod, with 60% humidity), the leaves were collected to analyze the malondialdehyde (MDA) content, soluble sugar content, free proline levels, and relative electric conductivity (REC). Leaves collected 2 weeks after chilling treatment were used to detect the in situ accumulation of superoxide radical (O2-). Additionally, we collected leaves at selected time points for semi-quantitative reverse transcription-PCR (RT-PCR) analysis. Eventually, 20 days after chilling treatment, all plants were evaluated at 4 °C for 7 days to assess the chilling injury index. The results validated that the MYB80 gene was related to cold tolerance of tomato plants, and that silencing of the MYB80 gene reduced the cold resistance ability.