Background: Fatigue is a frequent complaint after stroke and may be associated with dependence in activities of daily living, decreased quality of life, increased institutionalization and mortality. Although fatigue severity scale (FSS) is the most frequently used scale in stroke, validation studies are scarce. Objectives: This study aimed to examine the psychometric properties of FSS in subjects with stroke. Methods: A total of 46 subjects with stroke who were admitted for rehabilitation and 52 control subjects who were admitted for local musculoskeletal problems were included. A comprehensive assessment including functional independence measure, Folstein Mini-Mental State Examination, Hospital Anxiety and Depression Scale (HADS), visual analog scale for fatigue (VAS), FSS, and vitality subscale of 36-item Medical Outcomes Study Short-Form Health Survey (SF-36v) was conducted. FSS, VAS and SF-36v were repeated 7 days later. Results: FSS demonstrated excellent internal consistency in subjects with stroke (Cronbach’s alpha: 0.928). There was a moderate correlation between FSS and SF-36v (r = −0.498, p < 0.001). FSS was weakly correlated with HADS anxiety (r = 0.310, p = 0.041) and HADS depression (r = 0.334, p = 0.027). Test-retest reliability of SF-36v (ICC: 0. 746, CI: 0.518–0.866), VAS (ICC: 0.829, CI: 0.671–0.911) and FSS (ICC: 0.742, CI: 0.512–0.863, p < 0.001) was good. ICC values for individual items of FSS were good except for item 6. Conclusions: FSS is a valid and reliable scale to measure fatigue in stroke. FSS is not sensitive to differentiate fatigue in stroke from the control subjects with orthopedic problems with similar age and gender.