To explore candidate genes of tobacco important traits and provide insights of space mutagenesis, we conducted the whole genome re-sequencing (30x coverage) in tobacco mutant NC89-M that was generated by space mutagenesis, and wild-type NC89. In comparison to the reference genome, 1880413 single nucleotide polymorphisms (SNPs), 398922 Indels and 4969 structure variations (SVs) were observed in NC89, while 1876219 SNPs, 120 Indels and 42 SVs were detected in NC89-M. A total of 271655 SNPs and 23450 Indels differed between NC89-M and NC89, which resulted in sequence modifications on 8378 and 2156 genes, respectively. SNPs were often observed in mutant, suggesting that single nucleotide mutation is predominant using spare mutagenesis. The ratio of conversion type to transversion type is 2.053. It is proved that the single-base mutations are the main mutation type in the tobacco genome induced by space and conversion type is the main mutation type among the single base mutation. The number of insertion was significantly more than that of the deletion. It is proved that the insertion is the main mutation type in Indel caused by space mutagenesis. Among the SV, space mutagenesis mainly results in five types of structural variation including insertion, deletion, inversion, intra-chromosomal translocation and inter-chromosomal translocation. KEGG annotation of mutant genes indicated that the number of mutant genes in the metabolic pathway and biosynthesis of secondary metabolites are the highest. Gene function annotation indicated that several functional genes were modified in mutant, including MADS-box genes that control flowering time, KNOX1 gene that regulates development of lateral organ and leaf edge shape and also the genes that associate to the synthesis of terpenoids.