PurposeTo perform a meta-analysis to compare and critically evaluate the short and medium-term clinical outcomes of peroneus longus tendon autograft and hamstring tendon autograft in anterior cruciate ligament (ACL) reconstruction. The study further evaluates the viability of the PLT autograft as a non-inferior alternative to the conventional HT autograft.MethodWe independently searched the following electronic databases: PubMed, Cochrane Library, Embase, and Web of Science for all relevant articles from inception to March 2025, in only the English language. Clinical outcomes included: IKDC score, Lysholm score, Tegner score, Tegner-Lysholm score, Visual-Analog-Scale (VAS) score, American Orthopaedic Foot and Ankle Society (AOFAS) score, Modified Cincinnati score, graft diameter, thigh circumference, graft harvest time, Knee laxity & stability (Lachman test, Pivot Shift test, and Anterior Drawer test), and Donor-site morbidity. Funnel plots were used to detect publication bias. All statistical analysis was performed using R (version 4.1.3).ResultsA total of 25 studies, 9 RCTs, and 16 cohort studies containing 1944 patients were included in this analysis. No statistically significant differences were observed between PLT and HT autografts in functional outcomes, including Tegner scores, Lysholm scores, Tegner-Lysholm scores, VAS scores, or objective measures of knee stability and laxity across all follow-up periods. Notably, the PLT group demonstrated superior outcomes compared to the HT group, including significantly larger graft diameter, reduced graft harvesting time, improved Modified Cincinnati Scores at both 12- and 24-month follow-ups, and lower donor-site morbidity rates. Despite significantly higher IKDC scores in the PLT group at 6 months postoperatively, no significant differences were observed at 12-, and 18, 24, or 36-month follow-up. Conversely, PLT groups had slightly lower AOFAS scores at 12 or 24 months follow-up than the HT group.ConclusionThe PLT autograft demonstrates comparable clinical outcomes to HT autograft in ACL reconstruction while offering distinct advantages, including significantly larger graft diameter, reduced harvest time, and lower donor-site morbidity. These benefits, coupled with equivalent postoperative functional results, position the PLT autograft as a promising alternative to traditional HT autograft. Long-term follow-up and higher-quality studies are needed to support our results and further investigate the differences between PLT autograft and HT autograft.Level of evidenceLevel III.