Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disease characterized by chronic, profound, disabling, and unexplained fatigue. A variety of studies have been performed to establish objective biomarkers of the disease, including positron emission tomography (PET) molecular imaging and neuro-functional imaging using magnetic resonance imaging (MRI) and magnetoencephalogram (MEG). In this chapter, we summarize the results from PET, MRI, and MEG imaging. Regional cerebral blood flow and glucose utilization rates are decreased in patients with ME/CFS as compared with age- and sex-matched healthy subjects. Acetyl-L-carnitine uptake into the releasable pool of glutamate and serotonin transporters densities are decreased in a few specific brain regions, mostly in the anterior cingulate in the patients. Although it is hypothesized that brain inflammation is involved in the pathophysiology of ME/CFS, there was no direct evidence of neuroinflammation in patients. Our recent PET study successfully demonstrated that neuroinflammation is present in widespread brain areas in ME/CFS patients, and is associated with the severity of neuropsychological symptoms. Evaluation of neuroinflammation in patients with ME/CFS may be essential for understanding the core pathophysiology, as well as for developing objective diagnostic criteria and effective medical treatments for ME/CFS. By using specific neurological features of these patients such as prefrontal cortical atrophies and the over-guarding phenomenon were found using MRI and functional MRI, respectively. We here describe related pathophysiological findings and topics in order to aid in the development of future therapies for ME/CFS patients.