A gold standard measurement does not exist to assess fatigue symptoms across patient populations. Current tools only consist of self-report measures that may not assess fatigue objectively. The National Institutes of Health-Brief Fatigue Inventory (NIH-BFI) is a clinician-administered instrument developed as a more objective assessment of fatigue symptoms. This study assessed the NIH-BFI's validity in diverse clinical populations, which included patients diagnosed with a mood disorder, Parkinson's disease, cancer, and healthy controls. Results suggest good criterion-related and convergent validity for the NIH-BFI. Results also indicate significant differences in fatigue severity between cohorts. Moreover, the data also suggest significant differences among groups in depression, anxiety, pain catastrophizing, sleep quality, global mental and physical health, and cognitive functioning. This study proposes that the NIH-BFI is a valid clinician-administered measure of fatigue that can be administered in multiple clinical populations.