OBJECTIVE To acquire MRI diffusion data (apparent diffusion coefficient [ADC] and fractional anisotropy [FA] values, including separate measures for gray and white matter) at 3.0 T for multiple locations of the brain of neurologically normal dogs. ANIMALS 13 neurologically normal dogs recruited from a group of patients undergoing tibial plateau leveling osteotomy. PROCEDURES MRI duration ranged from 20 to 30 minutes, including obtaining preliminary images to exclude pathological changes (T2-weighted fluid-attenuated inversion recovery transverse and dorsal images) and diffusion-weighted images. RESULTS Globally, there were significant differences between mean values for gray and white matter in the cerebral lobes and cerebellum for ADC (range of means for gray matter, 0.8349 X 10−3 s/mm2 to 0.9273 X 10−3 s/mm2; range of means for white matter, 0.6897 X 10−3 s/mm2 to 0.7332 X 10−3 s/mm2) and FA (range of means for gray matter, 0.1978 to 0.2364; range of means for white matter, 0.5136 to 0.6144). These values also differed among cerebral lobes. In most areas, a positive correlation was detected between ADC values and patient age but not between FA values and patient age. CONCLUSIONS AND CLINICAL RELEVANCE Cerebral interlobar and cerebellar diffusion values differed significantly, especially in the gray matter. Information about diffusion values in neurologically normal dogs may be used to diagnose and monitor abnormalities and was the first step in determining the clinical use of diffusion imaging. This information provided an important starting point for the clinical application of diffusion imaging of the canine brain.