Previous studies have indicated differences in fiber quality parameters including fiber length and maturity within the canopy of cotton (Gossypium hirsutum L) plants. A 3-yr study was conducted to investigate the impact of within-plant variability on fiber length and maturity of upland cotton cultivars widely grown on the High Plains of Texas. Twelve upland cotton cultivars were grown in a randomized complete block design with three fi eld replications, in Lubbock, TX, during the 2012, 2013, and 2014 growing seasons. A box-picking harvesting method was used to individualize samples so that the within-plant variability of cotton fiber quality could be studied. Bolls harvested from different positions on the plants were ginned with a tabletop roller gin to minimize fiber damage. The lint from each fruiting position was blended with a supple needles laboratory blender to reduce within-sample variability while minimizing fiber breakage. Each sample collected from a diff erent fruiting position on the plant was tested on the Advanced Fiber Information System (AFIS) with three replications of 3000 fibers. The results indicated that cultivars such as FM 9170 B2F, NG 4111 RF, PHY 499 WRF, and FM 2484 B2F showed lower withinplant variability, while DP 1044 B2RF, PHY 367 WRF, and ST 5458 B2F showed relatively high within-plant variability for AFIS fiber length and fiber maturity. In conclusion, variations in within-plant fiber length and maturity among upland cotton cultivars could be a potential source of variability for breeding programs aimed at improving fiber quality.