Cotton fibres develop over four stages: initiation, elongation, secondary-wall thickening, and maturation. They develop a significant crystalline structure during the secondary wall thickening stage of development. Cotton fibres were harvested from 17 days to 60 days after flowering (dpa). Transmission Electron Microscopy (TEM), Interferometry, Attenuated Total Reflectance Fourier-transform Infrared (ATR-FTIR) spectroscopy, immunofluorescence labelling, and fluorescence spectroscopy were used to characterise the cotton fibres in different stages. It was found that, secondary wall thickening and micronaire remain fairly constant from 17 to 24 dpa, after that time significant change occurs until maturity. Maturity ratio increases as the fibres develop. Birefringence increases rapidly from 17 dpa to 26 dpa, then levels off up to 60 dpa. It is evident by comparing the lateral order index (LOI) and results from the binding of a crystalline-cellulose binding probe (CBM3a) that there is a significant increase in the degree of cellulose crystallinity from 17 dpa to 26 dpa. Hydrogen Bond Intensity (HBI) increased to 24 dpa and decreased from 24 to 40 dpa indicating significant changes in inter-molecular hydrogen bonds. From 40 to 60 dpa an increase of HBI was observed. It is concluded that during the maturation stage of cotton fibre development, water loss from lumen allows the cellulose chains to come closer together and to form intermolecular hydrogen-bonds. TEM, Interferometry, ATR-FTIR spectroscopy, and immunofluorescence labelling combined with fluorescence spectroscopy, were demonstrated to be useful techniques in quantifying physical changes in cotton fibres during development, offering advantages over traditional analytical techniques.