The secondary cell wall development of cotton fibers harvested at 18, 20, 24, 28, 32, 36 and 40 days after flowering was examined using attenuated total reflection Fourier transform-infrared (ATR FT-IR) spectroscopy. Spectra of deuterated cotton fibers did not demonstrate significant changes in their O–H stretching band shapes or positions during development. Only a progressive increase in O–H band intensity was observed. Results indicate that the highly crystalline cellulose component produced during secondary cell wall formation maintains the hydrogen bonding network observed for the primary cell wall. Other general changes were observed for the regular ATR spectra. A progressive intensity increase for bands assigned to cellulose I_β was observed during fiber development, including a marked intensity increase for vibrations at 1002 and 985 cm⁻¹. In contrast, C–O vibrational bands from dominant conformations observed at 1104, 1052, 1028 cm⁻¹ undergo a modest intensity increase during secondary cell wall development.