Abstract: Two tobacco genotypes, "118-3" and "Yueyan 98", were sequenced using restriction-site associated DNA (RAD) on the Illumina MiSeq PE300 platform, and 45 119 346 and 50 360 738 HQ clean reads were obtained in "118-3" and "Yueyan 98", respectively. Through sequences analysis, 25 343 SSR loci were identified in the reference genome sequences covered by 22 145 reads. Of these SSR loci, di-and tri-nucleotide repeat motifs were the two most abundant types, with the percentage of 71.59% in total SSR loci. Apart from mono-nucleotide type, the repeat counts of SSR motifs mainly ranged from 4 to 9. 23 346 primer pairs (92.12% of total SSR loci) were successfully designed using the Primer 3.0 software based on the bilateral sequences of SSR loci, and 323 (1.38%) of them showed polymorphism between the two genotypes according to the sequencing data. Fifty of the 323 primer combinations were randomly selected to verify their polymorphisms in 4 tobacco genotypes, the results showed that 76% of the primer pairs were polymorphic between the two sequenced genotypes, and the false positive rate was 24%; and the polymorphic rate in the other two genotypes was 20%. These results indicate that RAD sequencing could be used to develop a large number of SSR markers especially polymorphic SSR markers, which could provide basis for the development and application of SSR markers.