For effective use of tobacco germplasm resources resistant to TMV, and improve resistance breeding efficiency against TMV, the inheritance for TMV resistance and resistance gene in three flue-cured tobacco lines, CV87, FC8 and Kang88, were studied. CV87, FC8 and Kang88 were crossed with YunYan87 or ZhongYan100, respectively, and F1 plants were self-pollinated to produce F2 populations, then the TMV resistance of CV87, FC8, Kang88 and there F1, F2 populations were identified by inoculating TMV-C strain; In addition, N gene primers were used to detect N gene sequence in genomic DNA of CV87, FC8, Kang88, YunYan87 and ZhongYan100. Resistance identification showed that CV87, FC8, Kang88 and F1 group were immune to TMV, YunYan87 and ZhongYan100 were susceptible to TMV, and Chi-square （χ2） tests proved that the separation ratio of F2 groups were 3:1, in accordance with the dominant single gene inheritance; PCR results found that N gene was present in resistant lines, CV87, FC8, Kang88, did not exist in susceptible materials, YunYan87 and ZhongYan100. The results indicated that the TMV resistance of CV87, FC8 and Kang 88 were controlled by single dominant gene N.