Fourier-transform mid-infrared mapping and histochemical staining are used to reveal the location and relative importance of chemical components involved with the base of cotton fibers and their associated seed coat. These two complementary techniques are focused on the nature of the chemical components that hold cotton fibers at their bases to the seed coat and with other portions of seed coat fragments that are often found as part of the trash component of ginned cotton. Infrared results reveal waxes or long-chain alcohols adjacent to the shank of cotton fiber bases in the outer epidermal tissue in all regions of the cotton seed; uronate anions in the outer epidermis and pigment layers surrounding the bases of the fibers and strongly present in the upper palisade layer tissue of all seed regions; compounds containing carbonyl functionality, acids, and bases, at the juncture of the upper palisade and colorless layers; tannin or pretannin-type aromatic structures in the outer pigment layer and interior to the cells in the epidermal layer of all seed coat regions; and lignin-type aromatics in the “colorless” layer of all regions of the seed coat. The infrared eresults are complemented by staining with Oil Red O for waxes Ruthenium Red for pectins, acid phloroglucinol for lignins, and vanillin HCl for tannins. The results provide a better understanding of fiber-seed interactions that are important to the development of methods for improving the separation of cotton fibers from seed coats. In turn, this will help to avoid breaking fibers and pulling out seed coat fragments with the fibers during ginning.