Myalgic Encephalomyelitis/Chronic fatigue syndrome (ME/CFS) patients suffer from a variety of physical and neurological complaints indicating the central nervous system plays a role in ME/CFS pathophysiology. Diffusion tensor imaging (DTI) has been used to study microstructural changes in neurodegenerative diseases. In this study, we evaluated DTI parameters to investigate microstructural abnormalities in ME/CFS patients. We estimated DTI parameters in 25 ME/CFS patients who met Fukuda criteria (ME/CFSFukuda), 18 ME/CFS patients who met International Consent Criteria (ICC) (ME/CFSICC) only, and 26 healthy control subjects (HC). In addition to voxel-based DTI-parameter group comparisons, we performed voxel-based DTI-parameter interaction-with-group regressions with clinical and autonomic measures to test for abnormal regressions. Group comparisons between ME/CFSICC and HC detected significant clusters (a) with decreased axial diffusivity (p=0.001) and mean diffusivity (p=0.01) in the descending cortico-cerebellar tract in the midbrain and pons, and (b) with increased transverse diffusivity in the medulla. The mode of anisotropy was significantly decreased (p=0.001) in a cluster in the superior longitudinal fasciculus region. Voxel-based group comparisons between ME/CFSFukuda and HC did not detect significant clusters. For ME/CFSICC and HC, DTI parameter interaction-with-group regressions were abnormal for the clinical measures of information processing score, SF36 physical, sleep disturbance score, and respiration rate in both grey and white matter regions. Our study demonstrated that DTI parameters are sensitive to microstructural changes in ME/CFSICC and could potentially act as an imaging biomarker of abnormal pathophysiology in ME/CFS. The study also shows that strict case definitions are essential in investigation of the pathophysiology of ME/CFS.