Objective The aim of this study was to longitudinally determine the prognostic value of early postoperative quantitative 3T-MRI (magnetic resonance imaging) parameters of subchondral bone marrow for 2-year clinical and MRI outcome after matrix-associated autologous chondrocyte implantation (MACI) with autologous bone grafting (ABG) at the knee. Design Consecutive subjects who received MACI with ABG for treatment of focal osteochondral defects received MRI follow-up 3, 6, 12, and 24 months postoperatively. Quantitative MRI included bone marrow edema-like lesion (BMEL) volume measurements and single-voxel magnetic resonance spectroscopy (MRS; n = 9) of the subchondral bone marrow. At 2-year follow-up, morphological MRI outcome included MOCART (magnetic resonance observation of cartilage repair tissue) 2.0 scores. Clinical outcomes were assessed using Lysholm scores. Results Among a total of 18 subjects (mean age: 28.7 ± 8.4 years, n = 14 males) with defects at the medial or lateral ( n = 15 and n = 3, respectively) condyle, mean BMEL volume decreased from 4.9 cm3 at 3 months to 2.0 cm3 at 2-year follow-up ( P = 0.040). MRS-based bone marrow water T2 showed a decrease from 20.7 ms at 1-year follow-up to 16.8 ms at 2-year follow-up ( P = 0.040). Higher BMEL volume at 6 months correlated with lower 2-year Lysholm (R = −0.616, P = 0.015) and MOCART 2.0 scores (R = −0.567, P = 0.027). Larger early postoperative BMEL volumes at 3 months (R = −0.850, P = 0.007) and 6 months (R = −0.811, P = 0.008) correlated with lower MRS-based unsaturated lipid fractions at 2-year follow-up. Furthermore, patients with early postoperative bony defects showed worse MOCART 2.0 ( P = 0.044) and Lysholm scores ( P = 0.017) after 24 months. Conclusion Low subchondral BMEL volume and optimal restoration of the subchondral bone at early postoperative time points predict better 2-year clinical and MRI outcomes after MACI with ABG.