In this study, in order to screen disease-resistant tobacco germplasms, forty six different type sources of tobacco germplasms were selected as experiment materials. Field observation and molecular identification were used to screen and measure these tobacco germplasms’ disease resistance ability to TMV. The results of field investigation showed that 14 moderate resistant tobacco germplasms, 8 moderate susceptible germplasms and 2 susceptible ones were screened out from 24 unidentified tobacco geramplasms, while 22 tobacco geramplasms’ resistance to TMV characteristics are basically the same as the previous results. Further, by means of specific primers of TMV-resistant gene (CN), specific fragments were amplified in 36 tobacco geramplasms’ genomic DNA. Taken together, it showed that 62.9% tobacco germplasms which have CN gene or homologous gene showed moderate resistant or highly resistant to TMV by field identification, and 83.3% tobacco geramplasms which have CN gene or homologous gene showed moderate resistant or highly resistant to TMV by molecular identification, and by combining both field and molecular methods, 3 moderate susceptible tobacco geramplasms and 11 moderate resistant ones were identified.