Tobacco vein banding mosaic virus (TVBMV) is of increasing importance in tobacco production. Knowledge of the genetic structure and variability of the virus population is vital for developing sustainable management. In this study, 24 new TVBMV isolates from Sichuan Province together with 46 previous isolates were studied based on their coat protein sequences. Two distinguishable clades were supported by phylogenetic analysis. The summary statistics PS, AI and MC showed a strong TVBMV-geography association between the isolates from Southwest China (SW) and Mainland China (MC). Further analysis indicated that the spatial genetic structure of TVBMV populations is likely to have been caused by natural selection. Phylogeographic analysis provided strong support for spatial diffusion pathways between the Southwest and Northwest tobacco-producing regions. The TVBMV CP gene was found to be under negative selection, and no significant positive selection of amino acids was detected in the SW group; however, the isolates of the MC group experienced significant positive selection pressure at the first and third amino acid sites of CP. This study suggests that natural selection and habitat heterogeneity are important evolutionary mechanisms affecting the genetic structure of the TVBMV population.