Background: Diffusion weighted imaging (DWI) has useful diagnostic and predictive value in the assessment of glial tumors. Most studies evaluating the use of DWI in glioblastomas have done so in regions that overlap with abnormal T2/fluid attenuation inversion recovery (FLAIR) signal or contrast enhancement. Isolated DWI abnormalities, which do not overlap with contrast enhancing lesions, are less commonly described. Their relationship with the tumour, and implications for prognosis, are not well understood, though it has been speculated that these lesions may represent infiltrative tumour cells. To our knowledge, this is the first reported case where the presence of infiltrative tumour cells in an area of diffusion restriction has been confirmed via biopsy. Methods: A ring enhancing lesion and isolated DWI hyperintensity from a newly diagnosed patient were biopsied separately. Results: Pathological specimens from both targets were identified as glioblastoma (WHO Grade IV), negative for IDH-1 R132H mutation, with methylated MGMT promoter. Conclusions: In patients with glioblastoma, DWI hyperintensities distant from areas of abnormal T2/FLAIR or contrast enhancement can contain infiltrative tumour cells. The presence of isolated diffusion restriction may be a useful predictor of disease progression and prognosis but further investigation into the nature and behavior of isolated DWI lesions is required.