PURPOSE: The aim of this study was to develop, implement, and demonstrate a three-dimensional (3D) extension of the readout-segmented echo-planar imaging (rs-EPI) sequence for diffusion imaging. THEORY AND METHODS: Potential k-space acquisition schemes were assessed by simulating their associated spatial point spread functions. Motion-induced phase artifacts were also simulated to test navigator corrections and a real-time reordering of the k-space acquisition relative to the cardiac cycle. The cardiac reordering strategy preferentially chooses readout segments closer to the center of 3D k-space during diastole. Motion-induced phase artifacts were quantified by calculating the voxel-wise temporal variation in a set of repeated diffusion-weighted acquisitions. Based on the results of these simulations, a 2D navigated multi-slab rs-EPI sequence with real-time cardiac reordering was implemented. The multi-slab implementation enables signal-to-noise ratio-optimal repetition times of 1-2 s. RESULTS: Cardiac reordering was validated in simulations and in vivo using the multi-slab rs-EPI sequence. In comparisons with standard k-space acquisitions, cardiac reordering was shown to reduce the variability due to motion-induced phase artifacts by 30-50%. High-resolution diffusion tensor imaging data acquired with the cardiac-reordered multi-slab rs-EPI sequence are presented. CONCLUSION: A 3D multi-slab rs-EPI sequence with cardiac reordering has been demonstrated in vivo and is shown to provide high-quality 3D diffusion-weighted data sets.