Diffusion tensor imaging (DTI) data at 43 mum isotropic resolution has been acquired on the intact adult mouse brain in 28-h scan time by using a streamlined protocol, including specimen fixation and staining, image acquisition, reconstruction, post-processing, and distribution. An intermediate registration of each component image is required to achieve the desired microscopic resolution. Multiple parameters have been derived, including fractional anisotropy, axial and radial diffusivity, and a color-coded orientation map of the primary eigenvector. Each DTI dataset was mapped to a common reference space to facilitate future standardized analysis. Fiber tracking has also been demonstrated, providing 3D connection information. This protocol to acquire high-resolution DTI data in a robust and repeatable fashion will serve as a foundation to quantitatively study mouse brain integrity and white matter architecture, at what we believe to be the highest spatial resolution yet attained.