Levels of genetic variation within 81 tobacco collection, including four populations (N.rustica L. 43 landrace, N.tabacum L. 13 landrace，10 bred and 15 introduced lines), were investigated using sequence related amplified polymorphism(SRAP). The results were as follows：(1)Totally 163 loci were produced with 123 polymorphic loci(75.5%) using 17 different primer combinations selected from 160 primers combinations and there were ten loci every primer combinations. A high level of genetic differentiation among populations was detected based on Nei’s genetic diversity analysis (0.41), Shannon’s information index analysis (0.57). （2） The genetic differentiation was different within the four populations: N.rustica L. landrace population〉N. tabacum L. population landrace > introduced population> bred population. (3) The cluster analysis indicated 81 tobacco germplasms were divided as N. rustica L. and N. tabacom L. Genetic difference between Flue-cured,、sun/air-cured,、cigar、oriental and burley were small. The genetic relationship among populations was studied by Nei unbiased genetic coefficient and Nei unbiased genetic distance, clustering analysis and principal coordinate analysis (PCO). The genetic distance was far between N.rustica L. landrace population and the other three N.tabacum L. populations, which indicated less N.rustica L. lines were used to tobacco breeding. The genetic coefficient was 0.99 between introduced population and bred population. This indicated that introduced germplasm were applied to tobacco breeding much more than landrace germplasm, so the genetic diversity of breeding parents was lower.