Wolfram syndrome (WFS) is inherited as an autosomal recessive disease with main clinical features of diabetes mellitus, optic atrophy, diabetes insipidus and deafness. However, various neurological defects may also be detected. The aim of this study was to evaluate aspects of brain structure and function using PET-CT (positron emission tomography and computed tomography) and MRI (magnetic resonance imaging) in pediatric patients with WFS. Regional changes in brain glucose metabolism were measured using standardized uptake values (SUVs) based on images of (18F) fluorodeoxyglucose (FDG) uptake in 7 WFS patients aged 10.1-16.0 years (mean 12.9±2.4) and in 20 healthy children aged 3-17.9 years (mean 12.8±4.1). In all patients the diagnosis of WFS was confirmed by DNA sequencing of the WFS1 gene. Hierarchical clustering showed remarkable similarities of glucose uptake patterns among WFS patients and their differences from the control group. SUV data were subsequently standardized for age groups <13 years old and>13 years old to account for developmental differences. Reduced SUVs in WFS patients as compared to the control group for the bilateral brain regions such as occipital lobe (-1.24±1.20 vs. -0.13±1.05; p = 0.028) and cerebellum (-1.11±0.69 vs. -0.204±1.00; p = 0.036) were observed and the same tendency for cingulate (-1.13±1.05 vs. -0.15±1.12; p = 0.056), temporal lobe (-1.10±0.98 vs. -0.15±1.10; p = 0.057), parietal lobe (-1.06±1.20 vs. -0.08±1.08; p = 0.058), central region (-1.01±1.04 vs. -0.09±1.06; p = 0.060), basal ganglia (-1.05±0.74 vs. -0.20±1.07; p = 0.066) and mesial temporal lobe (-1.06±0.82 vs. -0.26±1.08; p = 0.087) was also noticed. After adjusting for multiple hypothesis testing, the differences in glucose uptake were non-significant. For the first time, regional differences in brain glucose metabolism among patients with WFS were shown using PET-CT imaging.