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The effect of structure on the properties of cotton fibers is yet to be fully understood even after many years of research. This is due to the presence of convolutions that occur at various intervals in cotton fibers. An attempt was made in this investigation to remove these convolutions using liquid ammonia treatment. The optical and X-ray orientation angles of two varieties of G. hirsutum cotton fibers were investigated at various stages of maturity, and results were compared. An American upland variety was also studied. Four-hour treatment of cotton fibers in liquid ammonia at a temperature of −50 °C ensures a complete change of the lattice structure from cellulose I polymorph to cellulose III polymorph. The cellulose I lattice structure is restored by boiling it in distilled water for 24 h. X-ray diffractograms confirm these conversions. Mature fibers after treatments are devoid of convolutions and are rounded in appearance with no central lumen. The scanning electron micrographs revealed these morphological structures. A close correlation exists between the optical and X-ray orientation measurements and are both strongly dependent on fiber maturity. In all the varieties studied, a maturity ratio of at least 0.8 is required for a cotton fiber to be of commercial value, in terms of strength and durability The progressive build-up of both the primary and secondary walls as the fiber matures shows a gradual decrease in helix angles and, hence, an increase in the orientation of the fibrils, conforming to the constant pitch model. The effect of convolutions on both the optical and X-ray orientation angle is found to be higher than 10%.