: The aim of this study is to determine whether different playing positions in football influence muscle asymmetry, which is a common cause of injuries in football. This study aimed to determine the difference in the functional and lateral asymmetry of the knee joint muscles measured using tensiomyography (TMG) between football players of different playing positions. : This study included 52 professional football players (25.1 ± 4.7 years) divided into three groups according to their playing position: defenders-DF (N = 18), midfielders-MF (N = 15), and forwards-FW (N = 19). Functional and lateral symmetry were investigated by applying TMG on the knee joint muscles Rectus femoris, Vastus medialis, Vastus lateralis, Biceps femoris, and Semitendinosus of the right and left leg, and using the algorithm proposed by the manufacturer and previous studies. : The results of one and two-way ANOVA revealed no significant differences in functional (F = 0.596, = 0.555, on average) and lateral asymmetry (F = 0.888, = 0.497, on average) from the aspect of muscle contraction velocity and muscle stiffness between football players of different playing positions. : The study results show that the specificity of the playing position in football does not influence the functional and lateral asymmetry of the knee joint muscles. However, it has been shown that there is a high percentage of players with lateral (n = 14.5, 39.6%, on average) and specifically functional asymmetry (n = 27, 51.9%, on average).