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(1) Background: We aimed to compare gender differences in knee biomechanics and neuromuscular characteristics, and to determine the relationships between lower limb muscle pre-activations and knee biomechanics during a single leg drop landing, in order to identify riskier landing patterns to prevent injury and intervene properly. (2) Methods: Descriptive laboratory cross-sectional study on 38 healthy untrained subjects with low to moderate physical activity status. (3) Results: During the initial-contact phase of landing, females demonstrated greater peak vertical ground reaction force (GRF) normalized to body weight (49.12 ± 7.53 vs. 39.88 ± 5.69 N/kg; p < 0.001; Hedge’s g = 1.37), peak knee anterior reaction force normalized to body weight (0.23 ± 0.04 vs. 0.17 ± 0.05 N/kg; p < 0.001; Hedge’s g = 1.33), and decreased pre-activation of the semitendinosus (45.10 ± 20.05% vs. 34.03 ± 12.05%; p = 0.04; Hedge’s g = 0.67). The final regression equation was peak knee anterior reaction force = 0.024 + 0.025 (peak knee flexion moment) − 0.02 (semitendinosus-to-vastus lateralis pre-activation ratio) + 0.003 (peak vertical GRF) (R2 = 0.576, p < 0.001). (4) Conclusions: Overall, the data provided in this study support that a reduced semitendinosus-to-vastus lateralis pre-activation ratio predicted an increase in knee anterior reaction force and potentially an increase in ACL forces. Female non-athletes had gender-specific landing characteristics that may contribute to ACL injury. Future studies are warranted to consider more possible predictors of non-contact ACL injury.