Abstract: In order to clarify the pathogens that causing symptoms of yellowing spots and necrotic spots in tobacco from Chongqing tobacco-growing area and to develop a comprehensive control strategy, 18 leaf samples were collected from Fuling and Wuxi tobacco-growing areas and Beibei experimental fields. Specific primers were designed according to the gene sequence of the virus coat proteins, and RT-PCR amplification and BLAST sequence alignment analyses were conducted. The results showed that Tobacco Mosaic Virus (TMV) and Chilli veinal mottle virus (ChiVMV) were detected from the samples with yellowing spots and necrotic spots in Fuling tobacco-growing area, and the detection rates were 54.55% and 100% respectively. TMV and Potato Virus Y (PVY) were detected from the samples in Wuxi tobacco-growing area, and the detection rates were 100% and 66.67% respectively. ChiVMV was detected from the diseased leaf samples in Beibei tobacco-growing experimental fields, and the detectable rate was 100%. ChiVMV was detected from tobacco leaves in Chongqing for the first time.