We investigated DTI changes, potentially indicating alterations of microstructure and brain tissue integrity in 13 patients with probable progressive supranuclear palsy (PSP, Richardson syndrome) at stage III or less and 10 age-matched controls using a whole brain analysis of diffusion tensor imaging (DTI) data. DTI images were analyzed using tract-based spatial statistics, a hypothesis-free technique. Fractional anisotropy (FA), radial diffusivity (RD), and axial diffusivity (AD) were determined. In patients with PSP, significant increases in FA (P < 0.0001), an unspecific measure of microstructural tissue integrity, were found in the cerebellum and in the superior cerebellar peduncle bilaterally, in the fornix, the body of the corpus callosum and the olfactory region, when compared with age-matched healthy controls. Further, regional reductions in AD (P < 0.0001), an indicator of altered axonal integrity, were observed in the pons, the right substantia nigra and the cerebellar white matter bilaterally. Significant increases in RD (P < 0.0001), a potential measure of altered myelin integrity, occurred bilaterally in the superior cerebellar peduncle, the cerebellar white matter, the vermis of the cerebellum, the fornix, the body of the corpus callosum, and the olfactory region. RD values in the superior cerebellar peduncle discriminated patients with PSP and controls with high sensitivity (0.92) and specificity (1.0). The findings are supported by neuropathological studies. Our data suggest the usefulness of this clinically available new technique as a possible tool for differential diagnosis.