There is a continuous change in cell wall composition and organization during cotton fiber development. Cotton fiber strength correlates to the molecular weight (MW) and molecular weight distribution (MWD), and organization of cellulose chains in the secondary cell wall. These parameters change drastically during fiber development. This study reports on the MW, MWD, and organization of cellulose in cotton fibers harvested from two cotton cultivars of Gossypium hirsutum L., (Texas Marker-1 and TX55) at different levels of maturity. Fiber dissolution is necessary to estimate the molecular properties of cellulose. Cellulose in mature cotton fibers is larger in MW and highly crystalline and, therefore, poorly dissolves in common solvent systems. To facilitate the dissolution, fibers were first pretreated with 23% sodium hydroxide and then dissolved in a dimethylacetamide/lithium chloride solvent system. Gel permeation chromatography of dissolved fibers indicated that cellulose in both cultivars reaches its maximum MW around 30 days post anthesis. Fourier transform infrared microspectroscopy imaging in the transmission mode indicates changes in cellulose distribution in cotton fibers with fiber development. The distributions of infrared vibrations of cellulose at 897 (β-linkage of cellulose), 1161 (anti-symmetrical C-O-C stretching of cellulose), and 1429 cm⁻¹ (CH₂ scissoring of cellulose) provided information on cellulose deposition in intact cotton fibers.