Three types of cotton fibers of different varieties and exhibiting different maturities and wax contents were examined by inverse gas chromatography (IGC), at infinite dilution. In this study, the potential relationships between the microstructure and the surface properties of different cotton fibers are analyzed. This method is based on the analysis of adsorption of gaseous probes on solid surfaces. By measuring the retention time of probes into a column containing the fibers, thermodynamic surface characteristics of these fibers, in particular the dispersive component of their surface energy, and their surface morphological aspects, are determined. The IGC data are correlated with a surface physico-chemical analysis performed by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy.