Fatigue is a major issue in neurorehabilitation without a gold standard for assessment. The purpose of this study was to evaluate measurement properties of the five subscales of the self-report questionnaire the Dutch Multifactor Fatigue Scale (DMFS) among Danish adults with acquired brain injury. A multicenter study was conducted (N = 149, 92.6% with stroke), including a stroke unit and three community-based rehabilitation centers. Unidimensionality and measurement invariance across rehabilitation settings were tested using confirmatory factor analysis. External validity with Depression Anxiety Stress Scales (DASS-21) and the EQ-5D-5L was investigated using correlational analysis. Results were mixed. Unidimensionality and partial invariance were supported for the Impact of Fatigue, Mental Fatigue, and Signs and Direct Consequences of Fatigue, range: RMSEA = 0.07–0.08, CFI = 0.94–0.99, ω = 0.78–0.90. Coping with Fatigue provided poor model fit, RMSEA = 0.15, CFI = 0.81, ω = 0.46, and Physical Fatigue exhibited local dependence. Correlations among the DMFS, DASS-21, and EQ-5D-5L were in expected directions but in larger magnitudes compared to previous research. In conclusion, three subscales of the DMFS are recommended for assessing fatigue in early and late rehabilitation, and these may facilitate the targeting of interventions across transitions in neurorehabilitation. Subscales were strongly interrelated, and the factor solution needs evaluation.