The current study is the first installment of an effort to explore the secondary gene pool for the enhancement of Upland cotton (Gossypium hirsutum L.) germplasm. We developed advanced-generation backcross populations by first crossing G. hirsutum cv. Tamcot 2111 and G. barbadense cv. Pima S6, then independently backcrossing F(1) plants to the G. hirsutum parent for three cycles. Genome-wide mapping revealed introgressed alleles at an average of 7.3% of loci in each BC(3)F(1) plant, collectively representing G. barbadense introgression over about 70% of the genome. Twenty-four BC(3)F(1) plants were selfed to generate 24 BC(3)F(2) families of 22-172 plants per family (totaling 2,976 plants), which were field-tested for fiber elongation and genetically mapped. One-way analysis of variance detected 22 non-overlapping quantitative trail loci (QTLs) distributed over 15 different chromosomes. The percentage of variance explained by individual loci ranged from 8% to 28%. Although the G. barbadense parent has lower fiber elongation than the G. hirsutum parent, the G. barbadense allele contributed to increased fiber elongation at 64% of the QTLs. Two-way analysis of variance detected significant (P<0.001) among-family genotype effects and genotypexfamily interactions in two and eight regions, respectively, suggesting that the phenotypic effects of some introgressed chromosomal segments are dependent upon the presence/absence of other chromosomal segments.