Peroneus longus tendon can be a suitable alternative autograft for anterior cruciate ligament reconstruction (ACLR). The present study aimed to compare the clinical outcome and donor site morbidity in ACLR using peroneus longus tendon autograft versus hamstring tendon autograft. In a comparative cross-sectional study, ACLR was performed with quadrupled hamstring tendon autograft in 65 patients between 2017 and 2018, and in another group, peroneus longus tendon autograft was used for ACLR in 65 patients between 2018 and 2019. The same surgical technique, fixation method, and postoperative protocol were used in both groups. The knee functional outcomes were assessed according to the Lysholm score and the International Knee Documentation Committee scale at preoperative and at the end of at least 2 years after the procedure. Moreover, graft diameter was measured intraoperatively. Thigh circumference, the American Orthopedic Foot and Ankle Score (AOFAS), The Foot & Ankle Disability Index (FADI), and ankle range of motion (ROM) were used to evaluate ankle donor site morbidities. A total of 130 patients (65 patients in each group) were evaluated with a minimum follow-up of 2 years (range 24-31 months). Both groups showed no significant differences in clinical outcomes and knee stability. The peroneus longus graft diameter was significantly larger than the hamstring diameter (P<0.001). Assessment of AOFAS, FADI, and ankle ROM showed no apparent ankle joint dysfunction in the peroneus longus tendon group. Faster knee extensions, less anteromedial knee pain, and thigh hypotrophy were observed in peroneus longus tendon patients. Peroneus longus tendon autograft can be an appropriate autograft for ACLR due to its strength, larger graft diameter, and avoiding potential complications of hamstring autograft obtained from the knee region.