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A peroneus longus tendon autograft is used in many orthopaedic procedures and it is biomechanically comparable to a hamstring tendon autograft. Despite its potential, there are few studies that have evaluated the use of the peroneus longus tendon in ACL reconstruction. The aim of this study was to compare the clinical outcome and donor site morbidity of ACL reconstruction with hamstring tendon autografts versus peroneus longus tendon autografts in patients with an isolated ACL injury. Patients who underwent isolated single-bundle ACL reconstruction were allocated to two groups (hamstring and peroneus longus) and observed prospectively. Graft diameter was measured intraoperatively. Functional scores (IKDC, modified Cincinnati and Lysholm scores) were recorded preoperatively and 1 year after surgery. Donor site morbidities were assessed with thigh circumference measurements and ankle scoring with the AOFAS and FADI. Fifty-two patients (hamstring n = 28, peroneus n = 24) met the inclusion criteria. The peroneus longus graft diameter (8.8 ± 0.7 mm) was significantly larger than the hamstring diameter (8.2 ± 0.8 mm) (p = 0.012). There were no significant differences between the pre- and 1-year postoperative score between the hamstring and peroneus longus groups in the IKDC (n.s), modified Cincinnati (n.s), and Lysholm (n.s). The mean for the AOFAS was 97.3 ± 4.2 and for the FADI 98 ± 3.4 in the peroneus longus group, with a significant decrease in thigh circumference in the hamstring group (p = 0.002). Anterior cruciate ligament reconstruction with peroneus longus autografts produces a functional score (IKDC, modified Cincinnati, Lysholm) comparable to that of hamstring autografts at a 1-year follow-up, with the advantages of larger graft diameter, less thigh hypotrophy and excellent ankle function based on AOFAS and FADI scores. Prospective cohort study, Level II.