Abstract Background Trinity Evolution® (TE), a viable cellular bone allograft, previously demonstrated high fusion rates and no safety-related concerns after single-level anterior cervical discectomy and fusion (ACDF) procedures. This prospective multicenter clinical study was performed to assess the radiographic and clinical outcomes of TE in subjects undergoing two-level ACDF procedures. Methods In a prospective, multicenter study, 40 subjects that presented with symptomatic cervical degeneration at two adjacent vertebral levels underwent instrumented ACDF using TE autograft substitute in a polyetherethereketone (PEEK) cage. At 12 months, radiographic fusion status was evaluated by dynamic motion plain radiographs and thin cut CT with multiplanar reconstruction by a panel that was blinded to clinical outcome. Fusion success was defined by angular motion (≤4°) and the presence of bridging bone across the adjacent vertebral endplates. Clinical pain and function assessments included the Neck Disability Index (NDI), neck and arm pain as evaluated by visual analog scales (VAS), and SF-36 at both 6 and 12 months. Results At both 6 and 12 months, all clinical outcome scores (SF-36, NDI, and VAS pain) improved significantly (p