Total genomic DNA of 80 tobacco materials (originated from China, America, Poland, Zimbabwe, Japan, Bulgaria, Zambia, Terkey, Yugoslavia, India, the Soviet Union, Albania) were amplified by 69 polymorphic SSR markers screened from a total of 200 SSR primer pairsand the length polymorphism of the amplified fragment was analyzed using Biosystems 3500xl Genetic Analyzer. The results revealed that a sum of 503 polymorphic bands were amplified using 69 SSR primer pairs. And an average of 7.91 alleles were observed for each primer pair with average heterozygosity(Ho) 0.2764, average expected heterozygosity(He) 0.574 7. Shannon’s index was 1.082 6, Nei’s index was 0.571 1, coefficient of genetic differentiation was 0.141 6, gene flow was1.515 3 and genetic similarity coefficient ranged from 0.55-0.90. The results showed that the genetic diversity of 80 tobacco germplasm was relatively abundant, the genetic differentiation was 14.16% between populations, most sites were seriously deviating from the Hardy Weinberg equilibrium indicating a low heterozygosity and a low gene flow between populations.