The objective of this study is to understand genetic diversity about flue-cured tobacco, find valuable molecular markers associated with aroma constituents, and excavate excellent alleles and germplasms, which is beneficial for the study of molecular marker assisted breeding of tobacco aroma quality. In this study, 76 kinds of aroma constituents were identified from 60 kinds of flue-cured tobacco germplasms from two areas. Test population were amplificated using 390 pairs of polymorphic primers screened from 1914 SSR markers covering the whole genome. Nei’s genetic distance and neighbour-joining were adoped in cluster analysis. Based on the detection of population structure, we used the mixed linear models to analysis the relations between phenotype and genotype and finded some excellent alleles and germplasms. The 390 pairs of polymorphic primers displayed 928 allelic variation, the average of polymorphism information content (PIC) was 0.332, which ranged from 0.141-0.733, which showed high genetic diversity. The test group was dicided into two subgroups based on Nei’s genetic distance, which reflected the genetic relationship among tobacco cultivars. Genetic structure analysis mainly consist with Nei’s genetic distance analysis. Correlation analysis showed that: at the level of P < 0.05, 41 kinds of aroma constituents were associated with 56 kinds of SSR markers. At the level of P <0.01, there were 5 markers significantly associated with 6 kinds of aroma constituents including oxalic acid, myristate acid, a-terpineol, linalool, 2,4-Heptadienal and gamma-amylbutyrolactone, the percentage of phenotypic variation explained ranged from 12.54%-42.20%. Besides, excellent alleles about fourteen aroma constituents were finded, future study obtained some germplasms with more positive alleles: Jingyehuang, 9201, Danyu 2, Manwuxiang, Jinxing6007, Qinyan 95, et al. These results may provide useful information for molecular marker assisted breeding in tobacco quality breeding and screening of parents.