The analysis of association between genotypic markers and phenotypic traits allows identification of quantitative trait loci (QTLs), which can be applied through marker-assisted selection in breeding programs to improve the quality of crops. However, success in these applications depends primarily on the stability and dominance of the QTL. We previously identified a significant fiber length (FL) QTL on chromosome (Chr.) D11 based on the genome-wide association study (GWAS) of a multi-parent advanced generation inter-cross (MAGIC) population in upland cotton. In this report, we conducted mapping studies with two bi-parental populations to confirm the stability of the FL QTL on Chr. D11 and determine the magnitude of its effect on the fiber length phenotype. One of the F2 populations was developed from a cross between the longest fiber and the shortest fiber recombinant inbred lines (RILs) of the MAGIC population originally used for GWAS, whereas the second F2 population was created from a cross between two cotton lines Acala 1517–80 and JJ1145ne, which were not among the eleven MAGIC parental lines. The populations were grown in different environmental conditions. Genetic mapping of these populations confirmed the stability of the FL QTL on Chr. D11. The highest LOD scores of association with fiber length in both populations showed three SNP markers that resided within 360 kb of the QTL region on Chr. D11. Ten genes possessing non synonymous SNPs (nsSNPs) in their protein coding regions were identified in this region. RNAseq analysis detected activity in developing fiber tissue for seven of these candidate genes.