In the present study, a newly introduced bovine cross-linked collagen scaffold (test material) was investigated in vivo in an Achilles tendon defect model and compared to a commercially available porcine collagen scaffold (control material). In total, 28 male Sprague Dawley rats (about 400 g) were examined. The defined Achilles tendon defect of 5 mm of the right hind limb was replaced by one of the scaffold materials. After euthanasia, the hind limbs were transected for testing. Biomechanical evaluation was carried out via tensile testing (n = 8 each group, observation time: 28 days). Nonoperated tendons from the bilateral side were used as a control (native tendon, n = 4). For the histological evaluation, 12 animals were sacrificed at 14 and 28 days postoperatively (n = 3 each group and time point). Stained slices (Hematoxylin & Eosin) were evaluated qualitatively in terms of presence of cells and cell migration into scaffolds as well as structure and degradation of the scaffold. All transected hind limbs were additionally analyzed using MRI before testing to verify if the tendon repair using a collagen scaffold was still intact after the observation period. The maximum failure loads of both scaffold materials (test material: 54.5 ± 16.4 N, control: 63.1 ± 19.5 N) were in the range of native tendon (76.6 ± 11.6 N, p ≥ 0.07). The stiffness of native tendons was twofold higher (p ≤ 0.01) and the tear strength was approximately fivefold higher (p ≤ 0.01) compared to the repaired tendons with both scaffolds. Histological findings indicated that neither the test nor the control material induced inflammation, but the test material underwent a slower remodeling process. An overall repair failure rate of 48% was observed via MRI. The experimental data of the newly developed test material showed similar outcomes compared to the commercially available control material. The high repair failure rate indicated that MRI is recommended as an auxiliary measurement tool to validate experimental data.