(1) Background: The present study examined the isokinetic peak torque exerted by both knee extensors and flexors, anterior-posterior imbalance and the magnitude and direction of inter-limb asymmetry in professional and academy soccer players. (2) Methods: One hundred soccer players (professional=50, elite academy=50) volunteered to take part in this investigation. An isokinetic dynamometer was used to measure the knee extensor (quadriceps) and flexors muscles (hamstrings) torques of the limbs as well as inter-limb asymmetries – using a standard percentage difference equation. (3) Results: Professional players exhibited significantly greater (effect size [ES] = large) strength levels for the quadriceps and hamstrings under both testing conditions, professional players showed significantly higher (small to moderate) intra-limb ratio values for 60°·s-1 but not for the 300°·s-1 test condition, professional players showed significantly (small to moderate) lower inter-limb asymmetry values for all test conditions, with the exception of the hamstrings at 60°·s-1 and, the direction of asymmetry was poor to slight, indicating that limb dominance was rarely the same between groups. (4) Conclusions: This study shows that isokinetic assessments, i.e., peak torque exerted by both knee extensors and flexors and intra-limb ratio, and the subsequent inter-limb asymmetry, i.e., magnitude and direction, can differentiate between professional and academy soccer players.