A backcross-self population from a cross between Gossypium hirsutum and G. barbadense was used to dissect the molecular basis of genetic variation governing 15 parameters that reflect fiber length. Applying a detailed restriction fragment length polymorphism (RFLP) map to 3,662 BC(3)F(2) plants from 24 independently derived BC(3) families, we detected 28, nine, and eight quantitative trait loci (QTLs) for fiber length, length uniformity, and short fiber content, respectively. For eight, six, and two chromosomal regions containing quantitative trait loci (QTLs) for fiber length, length uniformity, and short fiber content (respectively), two-way analysis of variance showed a significant (P<0.001) among-family genotypic effect. A total of 13, two, and four loci showed genotype x family interaction, illustrating some of the complexities that are likely to be faced in introgression of exotic germplasm into the gene pool of cultivated cotton. Co-location of many QTLs for fiber length, length uniformity, and short fiber content accounted for correlations among these traits, while the discovery of many QTLs unique to each trait suggests that maximum genetic gain will require breeding efforts that target each trait (or an index including all three). The availability of DNA markers linked to G. barbadense QTLs identified in this and other studies promise to assist breeders in transferring and maintaining valuable traits from exotic sources during cultivar development.