Arthroscopic reconstruction of anterior cruciate ligament (ACL) surgical procedure using hamstring autograft is themost common surgery performed in the arena of sports medicine and arthroscopy. Most studies in literature are ambiguous regarding the fate of hamstrings based on function, regenerative potential, and cross-sectional area (CSA). The aim of this research study is analysis of the fate of hamstring tendons (both semitendinosus and gracilis) during the time course for determinants of regeneration and strength. Fifty patients who were operated for unilateral isolated ACL reconstruction from July 2015 to June 2018 were evaluated for the fate of harvested hamstring tendons which included the following: regeneration, cross-sectional area (CSA), strength, and insertion of regenerated hamstrings by isometric torque and isokinetic strength. MRI of knee was performed for both knees concerning the semitendinosus (ST), gracilis (G), Sartorius, biceps femoris, and medial head of gastrocnemius. Eighty-four percent men and 16% women within a mean patient age of 34 ± 4.12 years were evaluated and all 50 (100%) patients demonstrated hamstring regeneration by the MRI measurements at six months and at one year post-ACL reconstruction. The torque of isometric knee flexion measured in 60° was found to be remarkably lower in the ACL-reconstructed lower extremity compared to that of the contralateral limb (87.13 ± 20.18% of BW), at 90° (49.17 ± 15.09% BW), and at 105° (43.91 ± 13.17% BW), respectively (p < 0.01). However, at 30° flexion and 45° flexion, the difference was insignificant (116.48 ± 21.07% BW for 30° and 100.16 ± 25.12% BW for 45°). It was found that the properties of musculotendinous units of ST and G were significantly transformed after their harvesting for ACL reconstruction and these weaknesses contribute to the flexion deficit of knee in the deeper range of flexion in the operated limb. Therefore, approaches facilitating tendon regeneration and preservation must be warranted.