Abstract：Chromosome segment substitution lines (CSSLs) consist of a battery of near-isogenic lines and cover the entire genome of some crops. With the exception of one or a few homozygous chromosome segment transferred from a donor parent, the remaining genome of each CSSL line is the same as the recipient parent. It is an ideal material for QTL mapping. In the present study, we developed a set of CSSLs that constituted of 182 lines, using G. hirsutum CCRI8 as the recipient parent and G. barbadense Pima90-53 as the donor parent, through molecular assisted-selection in BC3S1-3 generations. The genetic length of the substituted segments covered 19957.8cM with an average segment of 21.0cM, 4.7 times the total genetic length of upland cotton (4168.7cM). The substituted segments of each line varied in length, ranging from 0.7cM in the shortest segment to 83.2cM in the longest. The number of substituted segments ranged from 1 to 11. Fiber quality of the CSSLs was nearly normally distributed, and a part of the lines have been obviously improved in fober properties compared to CCRI8. These materials provide new genetic resources for further QTL mapping, as well as upland cotton fiber quality improvement.