Levels of genetic variation within 78 tobacco germplasm resistance to PVY, including three populations (Flue-cured 18, sun-cured 38, N.rustica L. 22), were investigated using simple sequence repeats (SSR). The results were as follows：(1)Totally 108 loci were produced with 96 polymorphic loci (88.89%) using 45 different primer combinations selected from 960 primers combinations. A high level of genetic differentiation among populations was detected based on Nei's genetic diversity analysis (0.3766), Shannon's information index analysis (0.5821) and the genetic similarity coefficient variation range（0.0454-0.9973）.(2) The genetic differentiation was different within the three populations: Sun-cured＞N. rustica L.＞Flue-cured. Compared the genetic distances among three groups, the similarity of mahogany and flue-cured tobacco genetic is 0.9840. (3) The cluster analysis indicated 78 tobacco germplasms were divided as N. rustica L. and N. tabacum L. There exists not a clear boundary line between Sun-cured and Flue-cured. The genetic relationship among populations was studied by Nei unbiased genetic coefficient and Nei unbiased genetic distance, clustering analysis. The genetic distance was far between N.rustica L. population and the other N.tabacum L. populations.