The development of molecular markers based on genome re-sequencing is a new method and trend. In this research three tobacco types, including two flue-cured tobacco cultivars (LY1306 line, Qinyan96), one sun-cured tobacco cultivar (Wanmao 3) as well as one Maryland tobacco cultivar (Wufeng 1), were re-sequenced on genomic nucleotide sequences. Combined with the genomic sequence of cultivars K326 and TN90, which have been sequenced and released in the National Center for Biotechnology Information (NCBI), the insertion and deletion (InDel) sites and single nucleotide polymorphisms (SNP) sites of these four re-sequenced tobacco cultivars were analysed. Seven simple sequence repeats (SSR) candidate alleles were identified from the genomic analysis that were supposed to be polymorphistic among these cultivars. Five of them were confirmed to be applicable after amplifying the fragments from the SSR allele sites, respectively. Verification PCR tests carried out on ten tobacco cultivars, including different tobacco types, revealed that they can be used to classify the tobacco cultivars or the tobacco types. Three of the SSR sites, including NW-015889872.1, NW-015854676.1 and NW-015890969.1, were shown to be very effective for the classification of flue-cured, Burley and Maryland tobacco types, as well as sun-cured tobacco types.