The study aimed to determine the influence of functional asymmetry on the kinematic parameters of the 50 m run and its significance in shaping maximum speed in sprinters at various sports levels. The analysis included 18 Polish sprinters (elite: ≤ 10.40 s, sub-elite: ≤ 11.10 s per 100 m) who performed four 50 m runs with 5-minute breaks. Kinematic parameters were recorded using the OptoJumpNEXT system and WittyGate photocells. The fastest and slowest run of each athlete was selected for analysis. The results showed that kinematic asymmetry has a significant impact on sprint performance. Elite sprinters had less asymmetry in stride length, frequency, and ground contact time, which correlated with better results. The key findings indicate that in the acceleration phase (0–20 m), stride length and contact time symmetry were crucial, while in the maximal speed phase (20–50 m), the symmetry of stride frequency was important. A higher sports level was associated with a more optimized running technique, as evidenced by lower kinematic asymmetry. The results suggest that minimizing kinematic asymmetry may be a crucial factor in optimizing the sprinting technique and enhancing performance, offering practical insights for coaches and athletes and empowering them to make informed decisions in their training programs.