Two generations(BC4F3 and BC4F3:4) of chromosome segment substitution lines (CSSLs), which were derived from a cross of CCRI45 (Gossypium hirsutum L. ) as the recipient parent and Hai 1 (Gossypium barbadense L.) as the donor parent, were used to evaluate the phenotypic traits related to fiber yield and quality . The results showed that the average value of all traits in two generations were near to those of recurrent parent CCRI 45, but there were still abundant genetic variation among the individuals, and some of them had better yield and fiber quality than CCRI45, in which 20lines showed stability for fiber length with above 30.00 mm and fiber strength with above 31.0 cN/tex. These CSSLs with excellent trait genes from G. barbadense could further be used for cotton quality breeding.