N.tabacum germplasm Honghuadajinyuan, Wankouhongtuyan, Baihuaheiyan, Yunyan 87 and wild tabacco germplasm N.alata were used as materials in this study. The genetic integrity of different populations was investigated by SSR with constructing mixed DNA pool. The results showed that, with the scanning of whole-genome, none of polymorphic SSR primers were screened from 960 SSR primer pairs of Honghua dajinyuan,Wankouhongtuyan and Baihuaheiyan,but 3 polymorphic SSR primers of Yunyan 87 were detected. 6 polymorphic alleles were amplified from 80 plants, and.they could still be achieved when the population was reduced to 10 plants..So the population greater than or equal to10 plants can represent the genetic integrity of N.tabacum germplasm.11 pairs of primers were selected from the 608 pairs to analyze N.alata, 44 alleles were amplified from the 80 plants, and the polymorphic alleles were 19, according to the comparison of genetic diversity parameters, a sample of no less than 20 plants should be represent the genetic integrity of the population.