A set of chromosome segment substitution lines (CSSLs) of tobacco (Nicotiana tabacum L.) was first developed by molecular marker assisted selection (MAS) and successive backcrossing with Y3, the flue-cured tobacco germplasm with comprehensive traits as the recipient parent, and two common tobacco cultivars Beinhart1000-1 and K326 as the donor parents. The cigar tobacco cultivar Beinhart1000-1 carries a variety of resistance traits including black shank (both race 0 and race 1) and brown spot resistance, while the flue-cured tobacco K326 was a commercial cultivar with high quality. In 256 CSSLs, a total of 377 substituted segments derived from donor Beinhart1000-1 and K326 in the genetic background of Y3 were distributed on 24 linkage groups. Each CSSL contains only 1–5 substituted segments and length of the substituted segments ranged from 0.05 to 36.88 cM with an average of 7.75 cM. The total length of the overlapped substituted segments was 2922.57 cM, which was 2.61 times of the whole tobacco genome. The genome covered length was 1114.32 cM, with a covered ratio of 99.45% of the recurrent tobacco genome. The CSSLs constructed in this study are excellent genetic materials for gene mapping, quantitative trait locus (QTL) analysis of quantitative traits, developing varieties by marker assisted selection in Nicotiana tabacum L.