The location of transgenes inserted into a genome are important in genetic studies and breeding programs. We conducted linkage analysis between 2,4-D resistant transgenes and 14 morphological marker genes in upland cotton (Gossypium hirsutum L.). Two separate germlines that exhibited monogenic dominance for resistance to 2,4-D were selected for linkage analysis. Multiple marker lines T582 and T586 were crossed with the 2,4-D resistant lines. Their F1, F2, and backcross/testcross progeny were produced and evaluated for segregation of resistance to 2,4-D and the marker loci. Linkage was found between 2,4-D resistance and the naked seed-1 morphological marker (18–37 cM), for both lines. Only two-point linkage tests were possible, so the orientation on the chromosome with respect to the marker could not be determined. Tests with aneuploid stocks deficient for the short arm of chromosome 12 placed the gene in the long arm of chromosome 12.