Proper defoliation of cotton is critical to maximize both harvest efficiency and fiber quality. Increased levels of leaf grade or trash resulting from inadequate defoliation can lead to decreases in fiber quality and value. Inherent characteristics of cultivars, such as leaf pubescence levels and defoliation practices influence the efficacy of cotton defoliation. This study aimed to determine the impact of leaf pubescence and defoliation strategies on defoliation success and fiber quality in cotton. Treatments included a factorial of four cultivars and two defoliation treatments. Cultivars included two smooth-leaf cultivars and two cultivars with greater leaf pubescence ratings. Defoliation treatments included a standard program and an aggressive program with increased rates of the same defoliant mixture and the addition of a desiccant. There were few instances of a cultivar-by-defoliation strategy interaction, however, both cultivar and defoliation strategy had a significant effect on defoliation ratings. The aggressive defoliation treatment decreased defoliation and increased desiccation in all three locations but did not influence yield or fiber quality. In two of three locations, cultivars with higher leaf pubescence ratings resulted in increased leaf grades and HVI trash ratings compared with the smooth leaf cultivars. The results of this study suggest that the defoliation strategy can impact efficacy of defoliation, whereas leaf pubescence characteristics influence fiber quality parameters.