Recently, the potato virus Y (PVY) strains that could break down the resistance of va-genotype tobacco varieties emerged on cultivated tobacco constantly. In 2016, a va-resistance breaking-down PVY strain, which was named PVY-CJ, was isolated and was verified and identified in this study. Re-inoculation on the va-genotype tobacco varieties, multiple RT-PCR, segmented amplification of the whole PVY genome by RT-PCR, molecular phylogenetic analysis and the sequence alignment of VPg protein of PVY strains were adopted to confirm and identify the PVY-CJ strain. Results showed that all the three va-genotype tobacco varieties showed infection symptoms when they were inoculated with PVY-CJ inoculum. PVY-CJ was classified as a PVY^NTN strain according to the result of multiple RT-PCR. Sequence analysis showed that the whole genome of PVY-CJ contained an open reading frame of 9180 nt encoding a polyprotein of 3060 amino acids. The result of molecular phylogenetic analysis further classified PVY-CJ as a PVY^NTN strain. A substitution of lysine to glutamic acid happened on position 105 of the amino acid of VPg of the PVY-CJ strain according to the sequence alignment of PVY-CJ with other PVY^NTN strains. A PVY strain, which could break down the resistance of va-genotype tobacco varieties in mainland China, was reported and identified for the first time. It provided material for breeding new PVY-resistant tobacco varieties.