Background: Bone marrow (BM) lesion differentiation remains challenging, and quantitative magnetic resonance imaging (MRI) may enhance accuracy over conventional methods. We evaluated the diagnostic value and inter-reader reliability of Dixon-based signal drop (%drop) and fat fraction percentage (%fat) as adjuncts to existing protocols. Materials and methods: In this prospective two-center study, 172 patients with BM signal abnormalities underwent standardized 1.5-T MRI protocols, including Dixon sequences. Two musculoskeletal radiologists independently evaluated images and performed quantitative measurements of %drop and %fat. Final diagnoses were established through histopathology (n = 96) or imaging follow-up (n = 76). Diagnostic value was assessed using area under the receiver operating characteristic curve (AUROC), inter-reader reliability using Cohen’s κ coefficient. Results: The consensus optimal cutoff was for %drop ≤ 19.8%, yielding 87.2% accuracy, 95.3% sensitivity, and 73.8% specificity, and that for %fat was ≤ 18.3%, achieving 86.6% accuracy, 96.3% sensitivity, and 70.8% specificity. Both metrics showed high diagnostic performance (AUROC 0.824–0.863) and excellent inter-reader reliability (κ > 0.93, p < 0.001). Multivariate analysis identified %drop ≤ 19.8% and %fat ≤ 18.3% as the strongest independent predictors of malignancy, with odds ratio (OR) being 9.38 and 8.85, respectively (p < 0.001). Signal characteristics on Dixon sequences provided additional diagnostic value, with signal voids on fat-only images (OR 7.14) and high signals on water-only images (OR 5.46). Conclusion: Quantitative MRI Dixon imaging parameters demonstrated high diagnostic accuracy and excellent inter-reader reliability in differentiating benign and malignant BM lesions, supporting their implementation in clinical practice protocols as a reproducible adjunct to conventional MRI. Relevance statement: Quantitative Dixon MRI provides reproducible, noninvasive differentiation of bone marrow lesions with high diagnostic accuracy across anatomical sites, enhancing clinical decision-making with standardized thresholds while demonstrating excellent inter-center consistency. Key Points: Quantitative Dixon MRI thresholds of %drop ≤ 19.8% and %fat ≤ 18.3% were established as reliable predictors of malignancy in bone marrow lesions. Dixon metrics demonstrated superior diagnostic accuracy (86.6–87.2%), compared to conventional T1-weighted sequences (79.2%). Excellent inter-reader reliability (κ = 0.895–0.943) supports the reproducibility of quantitative Dixon MRI in clinical practice. © 2025 Elsevier B.V., All rights reserved.