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The aim of this study was to observe direct and joint effects of three factors (genotypes, ecological environmental conditions and the applied crop density) on the level of defoliation intensity and yield. Three inbred lines (G) of maize (G1–L217RfC, G2–L335/99 and G3–L76B004) were used in the study. The trials were performed in two years (Y) (Y1 = 2016 and Y2 = 2017) and in two locations (L) (L1 and L2) under four ecological conditions of the year–location interaction (E1–E4) and in two densities (D1 and D2) (50,000 and 65,000 plants ha−1). Prior to tasselling, the following five treatments of detasseling and defoliation (T) were applied: T1—control, no leaf removal only detasseling, T2–T5—removal of tassels and top leaves (from one to four top leaves). The defoliation treatments had the most pronounced effect on the yield reduction in G1 (T1–Tn+1… T5), p &lt, 0.05. The ecological conditions on yield variability were expressed under poor weather conditions (E3 and E4), while lower densities were less favorable for the application of defoliation treatments. The result of joint effects of factors was the lowest grain yield (896 kg/ha) in G3 in the variant E3D1 for T2 and the highest grain yield (11,389 kg/ha) in G3 in the variant E2D2 for T1. The smallest effect of the defoliation treatment was on the kernel row number (KRN).