Background Quantitative MRI is increasingly proposed in clinical trials related to dystrophinopathies, including Becker muscular dystrophy (BMD). Purpose To establish the sensitivity of extracellular volume fraction (ECV) quantification using an MR fingerprinting sequence with water and fat separation as a quantitative imaging biomarker of skeletal muscle tissue alterations in BMD compared with fat fraction (FF) and water relaxation time quantification. Materials and Methods In this prospective study, study participants with BMD and healthy volunteers were included from April 2018 until October 2022 (ClinicalTrials.gov identifier NCT02020954). The MRI examination comprised FF mapping with the three-point Dixon method, water T2 mapping, and water T1 mapping before and after an intravenous injection of a gadolinium-based contrast agent by using MR fingerprinting, from which ECV was calculated. Functional status was measured with use of the Walton and Gardner-Medwin scale. This clinical evaluation tool stratifies disease severity from grade 0 (preclinical; elevated creatine phosphokinase; all activities normal) to grade 9 (unable to eat, drink, or sit without assistance). Mann-Whitney U tests, Kruskal-Wallis tests, and Spearman rank correlation tests were performed. Results Twenty-eight participants with BMD (median age, 42 years [IQR, 34-52 years]; 28 male) and 19 healthy volunteers (median age, 39 years [IQR, 33-55 years]; 19 male) were evaluated. ECV was higher in participants with dystrophy than in controls (median, 0.21 [IQR, 0.16-0.28] vs 0.07 [IQR, 0.07-0.08]; P < .001). In muscles of participants with BMD with normal FF, ECV was also higher than in muscles of healthy controls (median, 0.11 [IQR, 0.10-0.15] vs 0.07 [IQR, 0.07-0.08]; P = .02). ECV was correlated with FF (ρ = 0.56, P = .003), Walton and Gardner-Medwin scale score (ρ = 0.52, P = .006), and serum cardiac troponin T level (ρ = 0.60, P < .001). Conclusion Quantitative MR relaxometry with water and fat separation indicates a significant increase of skeletal muscle extracellular volume fraction in study participants with Becker muscular dystrophy. Clinical trial registration no. NCT02020954 Published under a CC BY 4.0 license. Supplemental material is available for this article.