Background: Third-generation autologous chondrocyte implantation (ACI) is an established method for treatment of full-thickness cartilage defects in the knee joint. Subchondral bone marrow edema (BME) is frequently observed after ACI, with unknown pathogenesis and clinical relevance. Purpose: To investigate the occurrence and clinical relevance of BME after third-generation ACI in the knee joint during the postoperative course of 36 months. Study Design: Cohort study; Level of evidence, 3. Methods: A total of 38 circumscribed full-thickness cartilage defects in 30 patients were included in this study. All defects were treated with third-generation ACI (Novocart 3D). A standardized MRI examination was carried out after 1.5, 3, 6, 12, 24, and 36 months. Bone marrow edema was observed in 78.9% of defects over the postoperative course, with initial occurrence in the first 12 months. The size of the BMEs were determined according to their maximum diameter and were classified as small (<1 cm), medium (<2 cm), large (<4 cm), and very large (diffuse; >4 cm). Clinical outcomes in patients were analyzed by use of the International Knee Documentation Committee (IKDC) scoring system and a visual analog scale for pain. Results: There were 5.3% (n = 2) small, 28.9% (n = 11) medium, 34.2% (n = 13) large, and 10.5% (n = 4) very large BMEs. In a subgroup analysis, cartilage defects of the medial femoral condyle showed significantly higher frequency of BME than did patellar defects. Clinical scores showed significant improvements throughout the entire study course (P < .05). Clinical patient outcome did not correlate with presence of BME at any time period (P > .05). Conclusion: Midterm clinical results of the matrix-based third-generation ACI showed a substantial amount of BME over a 36-month follow-up, but this did not correlate with worse clinical outcome. Patients with femoral cartilage defects were more often affected than were those with patellar cartilage defects.