Background: Periaqueductal gray (PAG) is a substantial descending pain modulatory center, and previous voxel-based morphometry study confirmed the clusters with increased volume in PAG region in medication-overuse headache (MOH). The aim of this study is to investigate altered PAG volume in MOH using an automated PAG segment method to measure the true PAG volume. Methods: High resolution three-dimensional T1-weighted fast spoiled gradient recalled echo MR images were obtained from 22 patients with MOH and 22 normal controls (NC). PAG template was created based on ICBM 152 gray template, and the individual PAG was generated by applying the deformation field from structural image segment to the PAG template, and individual PAG volume was calculated. Results: There was a significant increased volume of PAG in MOH (0.366 +/- 0.005 ml) than that in NC (0.341 +/- 0. 005 ml)(P0.05). The area of receiver operating characteristic (ROC) curve was 0.845, and the cut-off of PAG volume was 0.341 ml with sensitivity 95.5% and specificity 63.6%. Conclusion: The present study demonstrated that the PAG volume gain was confirmed in MOH patients, and the automated individual PAG volume measure may be considered as a simple and effective imaging biomarker in MOH diagnosis.