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• The main effect and interaction effect among the flight parameters were discussed. • The flight parameters of UAVs have remarkable effects on the droplet distribution. • The influence of flight parameters on droplet distribution uniformity is limited. A common problem of unmanned aerial vehicles (UAVs) spraying is that fewer droplets could reach the lower layer, and the depositions among the spray width show a significant difference. However, ensuring the penetration and uniformity of the spray is particularly important in the cotton harvest aid applications. As a new spraying method, poor droplet penetration and uniformity of droplet distribution may lead to inconsistent defoliation among canopy rows and poor defoliation in the lower part of the cotton canopy. The operational parameters of UAVs have remarkable effects on the droplet distribution, which will improve the defoliation rate of cotton. Therefore, full factorial tests were conducted in two experimental locations in the Xinjiang cotton-producing region in 2018 and 2019. The selected operational parameters in 2018 included the spray volume (three levels: 8.7, 12, and 15 L/ha), droplet size Dv50 (three levels: 100, 150, and 200 μm), and flight height (three levels: 1, 2, and 3 m). Meanwhile, the operational parameters in 2019 included the spray volume (three levels: 18, 22.5, and 30 L/ha) and droplet size Dv50 (three levels: 100, 150, and 200 μm). The main effects and interaction effects among the operational parameters were analyzed and discussed. The spray penetration and uniformity of droplet distribution were compared separately. The results show that flight height, spray volume, and droplet size have significant effects on spray penetration. Lowering the flight height of the drone, increasing the spray volume and Dv50 of droplets can improve the distribution of droplets at the bottom of the cotton canopy. As for droplet distribution uniformity, with the increase of the application volume and decrease of the flight height, the uniformity of the droplet distribution becomes worse. The influence of flight parameters on the uniformity of droplet distribution is very limited. [ABSTRACT FROM AUTHOR]