Abstract: Simple sequence repeats(SSRs)of tobacco expressed sequence tags(EST)in public database were investigated by using computer program MISA(MIcroSAtellite).Up to 81757 SSRs were found in 133523 sequences and the average distance between SSRs was approximately 0.92 kb.Among them,hexanucleotide repeats(60.3%)were the most abundance,while the monomeric,trimeric,tetrameric,dimeric,and pentameric repeats are represented in decreasing proportions of 20.0%,11.0%,4.2%,2.8%,and 1.7%,respectively.The most abundant motif was A/T,AG,AAG and AAAT in monomeric,dimeric,trimeric,and tetrameric repeats,respectively.Whereas CG rich repeats are rarely found in the coding regions.The redundancy analysis indicated that no significant differences were observed between the redundant and non-redundant set of tobacco ESTs in individual SSR motifs.Ten pairs of primers flanking EST-SSR loci were designed to detect the polymorphism in seven tobacco cultivars.The analyses on denatured PAGE by silver staining confirmed the existence of polymorphism by these four pairs of primers of EB4,EB5,EB6,and EB8 among the seven tobacco cultivars.The SSR loci reported in this study are the first molecular DNA-based genetic markers developed from public ESTs database in tobacco.The EST-SSR markers will be useful information resource for germplasm characterization and crop improvement through genetic mapping and marker-assisted selection in tobacco.