Levels of genetic variation within 81 tobacco core 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) in total 163 loci were produced with 123 polymorphic loci (75.5%) using 17 different primer combinations selected from 160 primer combinations and there were ten loci for every primer combination. 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) a moderate genetic differentiation (Gst=0.39) was found among the populations, suggesting that 39% genetic variation existed among the populations. 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. tabacum L. Flue-cured, sun/air-cured, cigar, Oriental and Burley showed little genetic diversity. 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 is 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 breeding parents' genetic diversity was limited presently.