Abstract: In order to screen for Potato virus Y (PVY) resistant mutant of tobacco among EMS mutant library, seedling inoculation was used to screen for M2 PVY-resistant plants, and continuous inoculation was conducted to identify M3, and M4 and M5 lines with resistance to PVY. The eIF4E1 molecular marker test and full length cDNA sequencing were applied to analyze whether the PVYresistant mutations were associated with the eIF4E1 mutation or not. F1 populations were used for preliminary analysis of the genetic characteristics of the resistance. After artificial inoculation of 1800 M2 lines at the seedling stage, a flue-cured tobacco mutant resistant to PVY necrotic strains was obtained. Seedlings were inoculated with PVY MN isolates and PVY ZT-5 isolates. After 35 days post inoculated (dpi), the disease incidence of E9119-1Z M3 line was 56.3% while that of the WT control was 100%. The symptomless M3 plantlets were PVY negative when tested by ELISA. These primary results indicated that the E9119-1Z/M3 line had medium resistance to PVY. In the green house inoculation test the M4 and M5 lines descendent of E9119-1Z showed stable medium resistance to PVY necrotic strains MN isolate and ZT-5 isolate. The E9119-1Z-R14/M5 was named pvyr1. Analysis of pvyr1 hybrid F1 with WT control and with the va locus PVY-resistant NC55 line showed that the PVY resistance of pvyr1 was in accord with Mendelian model of recessive gene and is not allelic to the NC55 va locus. The eIF4E1 gene specific molecular markers tests and cDNA sequencing results indicated that the pvyr1 mutation is not related to the eIF4E1 mutation. Mutant pvyr1 can be used as a new PVY-resistant tobacco germplasm.