Abstract: The population structure and genetic diversity of tobacco provide a theoretical basis for identifying elite genes and improving varieties by breeders. Genetic diversity and population structure of 87 tobacco materials were evaluated using 41 polymorphic SSR markers screened from a total of 80 SSR primer pairs. The amplified fragment length polymorphism was detected by The Applied Biosystems 3500 xl Genetic Analyzer. The results revealed that a sum of 241 polymorphism sites were found using the 41 SSR primer pairs, with an average of 5.88 sites per primer pair. The genetic similarity coefficient ranged from 0.47-0.91, and the PIC averaged 0.63, ranging from 0.23-0.91. Results from the clustering analysis based on a model-based method indicated that when K=2, the largest delta K value, namely 87 tobacco materials can be divided into two groups (P1 and P2, with the P1 group further divided into five classes and the P2 group further divided into two classes. The genetic diversity of tobacco germplasm was abundant and the division of population structure was not completely related to germplasm type.