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This research investigates the influence of tibial rotations with knee flexion (KF) on the electromyographic (EMG) activities of hamstring muscles (HM) groups and the strength ratio between the medial and lateral rotation of the tibia. A cross-sectional design was employed to assess muscle activity, isometric strength and reliability. The research was conducted in a controlled laboratory environment. Thirty-six amateur male athletes were recruited as volunteers. The measures included peak muscle activity of the semitendinosus and biceps femoris long head, the knee flexors' isometric strength ratio and reliability. The isometric strength ratios of medial (MR) to lateral (LR) tibial rotations were 0.94 ± 0.17 at 90°, and 0.93 ± 0.10 at both 60° and 30° of KF angulation. Tibial position significantly influenced knee flexion strength as well as HM activity, irrespective of KF angulation. Specifically, biceps femoris activity increased by 33.6% in LR compared to MR, while semitendinosus activity increased by 22.6% in MR compared to LR. The Knee-Rotation test (KR-test) can be a valuable tool for evaluating both HM groups (ICC >0.87), and identifying the primary target for strengthening purposes during the injury prevention process. It provides insights for effective rehabilitation and training interventions. • Reliability was high (ICC >0.87) for the KR-test. • Tibial rotations affect the knee flexion isometric strength. • Hamstring muscle groups should be considered as two separate entities in an assessment perspective. • Lateral rotation constantly increases the biceps femoris muscle activity, but medial rotation does not statistically increases the semitendinosus muscle activity. • The isometric strength ratios of medial/lateral knee flexion strength were 0.94 ± 0.17 at 90° and 0.93 ± 0.10 at 60° and 30° of knee flexion angulation. [ABSTRACT FROM AUTHOR]