Abstract: One tobacco virus disease sample was collected from Guiding tobacco regions in Guizhou province at 2007 which showed typical PVYN strain symptom. After three successive inoculations on Lycium barbarum L. as necrotic lesion host of PVY, and PVY was purified and maintained on Nicotiana tabacum Samsun NN plant, and named Guiding-3. Three pairs of specific primers based on genome sequences of PVY registered in GeneBank were designed. After total RNA extraction, RT-PCR, product sequencing, the sequence of PVY Gduiing-3 isolate was analyzed. The result showed that the genome sequence of Guiding-3 (NCBI Access ID HM590405） contained 9 699 bases, which had only one ORF of 9 186 bases and encoded a polyprotein, including 3 061 amino acids. Through BLAST, we found that the sequence similarity between Guidng-3 and PVYN, PVYNTN, PVYN:O, PVYO, PVYNNP were 98%, 97%, 94%, 92%, 85%, respectively. Furthermore, RNA phylogenetic tree suggested that Guiding-3 had high homology with PVYN strain. The molecular variation of PVY was also discussed in this paper.