Simple sequence repeats(SSRs) in the whole genomes of three wild tobaccos-Nicotiana tomentosiformis,Nicotiana sylvestris and Nicotiana benthamiana downloaded from the China Tobacco Genome Database(CTGDB) was analyzed by the bioinformatics method.A total of 153 357,196 493 and 278 784 SSR loci were mined out in the 2.14 Gb,2.44 Gb and 2.59 Gb of the three wild tobaccos’ genomic sequences,and the average distance between SSR loci was approximately 13.94 kb,12.42 kb and 9.31 kb,respectively.Almost all the SSR loci were distributed in intron domains and UTR domains(especially the 5′-UTR).Di-nucleotide and tri-nucleotide repeats,totally accounting for more than 80%,were the main motif types and the di-nucleotide was the most abundance in all SSRs.The highest frequency and the most abundance of SSR motifs configuration were A(T)_n.Except the mono-nucleotide motif type,the repeat number of the motif types was between 3 and 10.Three hundred SSR primer pairs randomly synthesized from three wild tobacco genomes were subjected to PCR using 8 tobacco accessions as materials which belong to 5 different species of the genus Nicotiana and the results showed that all the primer pairs could produce clear,stable and target bands,of which 80 SSR primers were polymorphic.The results indicated that SSR loci in the closer genetic relationship among tobacco species were highly conserved and could be used in the following research.It is meaningful to develop SSR primer pairs from wild tobacco genomic data for the application of relative genetic analysis.