The developing cotton fibre is an excellent model to study cell wall biochemistry, structure and function as it is a single cell with an unusual capacity for cell elongation and cell wall synthesis. Mature cotton fibres are the purest form of cellulose in nature, nonetheless the primary and secondary cell walls contain non-cellulosic polysaccharides and glycoproteins that modulate fibre development and may ultimately determine fibre quality traits such as length, fineness and strength. A comparative analysis of the fibre cell wall changes during development was carried out on selected cultivars with distinct fibre properties from four domesticated species G. hirsutum, G. barbadense, G. herbaceum and G. arboreum. Significant differences were found between species regarding the developmental dynamics of non-cellulosic epitopes, the developmental pace in terms of cell wall adhesion and detachment through the cotton fibre middle lamella, the timing of the transition phase and the cellulose deposition rate. The analysis of monosaccharide linkage and composition, and polysaccharide epitope immunolocalization using monoclonal antibodies, evidenced for the first time the presence of the hemicellulosic polysaccharide mannan in the developing and mature cotton fibre. Moreover, high magnification images of the fibre cell wall showed novel localization of several non-cellulosic polysaccharides, such as the presence of arabinoxylan between cellulose layers in the secondary cell wall. In addition, very important developmental processes, such as degradation/modification of pectic galactan and highly methyl-esterified homogalacturonan were developmentally profiled. Finally, further analysis of fibre glycan biochemistry led to the discovery of the capacity of CtCBM3a to bind to xyloglucan in addition to crystalline cellulose.