Chromosome segment substitution lines (CSSLs), in which each line carries a single or a few defined chromosome segment of donor genome and has a pure genetic background from a recurrent genotype, is a fundamental approach to conduct the QTL mapping in order to improve the mapping precision. In the present study, variety ‘ZM36’ (Gossypium. hirsutum) and ‘Hai1’ (Gossypium. Barbadense) were used as recurrent and donor parent to produce a segment substitution lines populations, respectively. The results indicated that most of fiber quality traits, yield-related traits and verticillium wilt traits fit the normal distribution in the Kolmogorov-Smirnov test. In 133 lines of BC4S2:3 generation fiber length longer the recurrent parent has 37, micronaire lower than the recurrent parent has 58, Fiber strength over than the recurrent parent has 34. In 199 individuals of BC4S2:4 generation fiber length longer the recurrent parent has 37, micronaire lower than the recurrent parent has 36, Fiber strength over than the recurrent parent has 49. Fiber length had significantly positive correlation with fiber strength and negative correlation with micronaire, seed index had significantly positive correlation with boll weight and negative correlation with lint percentage, lint percentage had negative correlation with fiber length and strength.