Purpose To investigate the surgical outcomes of anatomical anterior cruciate ligament (ACL) reconstruction according to the graft isometry measured during surgery. Methods Electrical medical records of patients who underwent an arthroscopic ACL reconstruction through the transportal technique using hamstring tendon autograft between 2012 and 2016 were retrospectively reviewed. The patients were classiied into two groups based on the graft length change throughout the knee range of motion measured just before graft ixation (Group 1, graft length change ≤ 2 mm; Group 2, graft length change > 2 mm). Comparative analyses, including a noninferiority trial, were performed regarding the clinical scores, knee laxity, and radiographic parameters between the groups. Results A total of 67 patients were included in the study. The total change in the length of ACL graft throughout the knee range of motion was 1.4 ± 0.4 mm in Group 1 (range, 0.2-2.0 mm), and 3.0 ± 0.7 mm in Group 2 (range, 2.2-5.0 mm). Group 1 showed a relatively high (proximal) femoral tunnel and shallow (anterior) tibial tunnel compared to Group 2 (P < 0.001 and P = 0.028, respectively), but there were no apparent diferences in the macroscopic view. There were no statistically signiicant diferences in the clinical outcomes between groups at 2 years after surgery, which satisied the non-inferiority criterion of Group 1 in terms of clinical scores and knee laxity compared to Group 2. Conclusion The surgical outcomes of anatomical ACL reconstruction in patients with non-isometric ACL graft were not inferior in terms of clinical scores and knee laxity, compared to those with nearly-isometric ACL graft. The graft tunnel placement in the isometric position during anatomical ACL reconstruction, which is technically challenging in the clinical setting, is not a crucial factor in terms of clinical outcomes. Level of evidence Level IV.