Abstract: The purpose of our study is screening out suitable SSR core primers which can accurately evaluate germplasm genetic diversity and genetic relationship of flue-cured tobacco. In this study, 2317 pairs of SSR primers distributed in 24 chromosomes were initially screened on 5 flue-cured tobacco cultivars with distant genetic relationship, and 70 pairs of primers were screened out. Then 20 flue-cured tobacco cultivars with different geographical origins were chosen to screen the initial selected primers. Finally, a total of 24 pairs of SSR core primers were identified. The genetic diversity of 20 flue-cured tobacco cultivars was nalyzed using these core markers. As a result, 85 polymorphism bands were acquired. The average PIC (Polymorphism information content) value is 0.52 and the mean number of alleles detected for each locus was 3.54. These 20 tobacco cultivars were conducted on both genetic diversity analysis and PCoA (Principal Coordinate Analysis) to demonstrate the effectiveness of this 24 core primers. These two methods exhibited similar phylogenesis among the tested cultivars and the results of molecular clustering largely agreed with the pedigree relationship. The results showed that the core primers were effective and accurate, and could be applied to flue-cured germplasm identification and genetic diversity study in tobacco.