Abstract: Single Nucleotide Polymorphisms (SNPs) are the most widely used molecular markers in the genomics field. In this study, high throughput SNPs were developed through re-sequencing of ten tobacco varieties using Restriction-site Associated DNA (RAD) technology. The sequencing clean data of 44.33 Gb was obtained, with an average genome coverage of 1.01 X. 291,770 SNPs were identified and the average spacing between adjacent SNPs was 10.066±29.801 kb. The SNPs cover the whole genome, but the distribution density of SNPs varies with the location of chromosomes. There is a long range of SNP-dense area in the upper arm of the 17th chromosome. Functional annotation showed that transition is the main type of SNP mutation and 45,049 SNPs were found in the gene regions. Based on the SNPs population genotyping information, genetic distance between the tested varieties was calculated, and the results showed that the average genetic distance is 0.29 and the genetic distance between Taiyan 8 and other varieties is the farthest. The results will provide scientific basis for tobacco QTL mapping, candidate gene discovery and parental group matching.