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Background: The biomechanical and tendon-bone incorporation properties of allograft-augmented hybrid grafts for anterior cruciate ligament (ACL) reconstruction compared with traditional autografts are unknown. Hypothesis: Using an autograft for ACL reconstruction yields better results on biomechanical testing, radiographic analysis, and histological evaluation versus using a hybrid graft. Study Design: Controlled laboratory study. Methods: A total of 66 adult male Sprague Dawley rats underwent unilateral ACL reconstruction with an autograft (AT group; n = 33) or a hybrid graft (HB group; n = 33). The grafts used in both groups were harvested from the peroneus longus tendon and were fixed by suturing to the surrounding periosteum. Samples were harvested for biomechanical testing, micro–computed tomography (CT), and histological evaluation at 4, 8, and 12 weeks postoperatively. Bone tunnels on the femoral and tibial sides were divided into 3 subregions: intra-articular (IA), midtunnel (MT), and extra-articular (EA). A cylinder-like volume of interest in the bone tunnel and a tubular-like volume of interest around the bone tunnel were used to evaluate new bone formation and bone remodeling, respectively, via micro-CT. Results: In the AT group, there were significantly higher failure loads and stiffness at 8 weeks (failure load: 3.04 ± 0.40 vs 2.09 ± 0.54 N, respectively; P =.006) (stiffness: 3.43 ± 0.56 vs 1.75 ± 0.52 N/mm, respectively; P <.001) and 12 weeks (failure load: 9.10 ± 1.13 vs 7.14 ± 0.94 N, respectively; P =.008) (stiffness: 4.45 ± 0.75 vs 3.36 ± 0.29 N/mm, respectively; P =.008) than in the HB group. With regard to new bone formation in the bone tunnel, in the AT group, the bone volume/total volume (BV/TV) was significantly higher than in the HB group on the tibial side at 8 weeks (IA: 22.21 ± 4.98 vs 5.16 ± 3.98, respectively; P <.001) (EA: 19.66 ± 7.19 vs 10.85 ± 2.16, respectively; P =.030) and 12 weeks (IA: 30.50 ± 5.04 vs 17.11 ± 7.31, respectively; P =.010) (MT: 21.15 ± 2.58 vs 15.55 ± 4.48, respectively; P =.041) (EA: 20.75 ± 3.87 vs 10.64 ± 3.94, respectively; P =.003). With regard to bone remodeling around the tunnel, the BV/TV was also significantly higher on the tibial side at 8 weeks (MT: 33.17 ± 8.05 vs 15.21 ± 7.60, respectively; P =.007) (EA: 25.19 ± 6.38 vs 13.94 ± 7.10, respectively; P =.030) and 12 weeks (IA: 69.46 ± 4.45 vs 47.80 ± 6.16, respectively; P <.001) (MT: 33.15 ± 3.88 vs 13.76 ± 4.07, respectively; P <.001) in the AT group than in the HB group. Sharpey-like fibers had formed at 8 weeks in the AT group. A large number of fibroblasts withdrew at 12 weeks. In the AT group, the width of the interface was significantly narrower at 4 weeks (85.86 ± 17.49 vs 182.97 ± 14.35 μm, respectively; P <.001), 8 weeks (58.86 ± 10.99 vs 90.15 ± 11.53 μm, respectively; P =.002), and 12 weeks (42.70 ± 7.96 vs 67.29 ± 6.55 μm, respectively; P =.001) than in the HB group. Conclusion: Using an autograft for ACL reconstruction may result in improved biomechanical properties and tendon-bone incorporation compared with a hybrid graft. Clinical Relevance: Augmenting small autografts with allograft tissue may result in decreased biomechanical performance and worse tendon-bone incorporation, increasing the risk of graft failure. [ABSTRACT FROM AUTHOR]