Magnetic Resonance Imaging (MRI) of the rodent brain at ultra-high magnetic fields (> 9.4 Tesla) offers a higher signal-to-noise ratio that can be exploited to reduce image acquisition time or provide higher spatial resolution. However, significant challenges are presented due to a combination of longer T1 and shorter T2/T2* relaxation times and increased sensitivity to magnetic susceptibility resulting in severe local-field inhomogeneity artefacts from air pockets and bone/brain interfaces. The Stejskal-Tanner spin echo diffusion-weighted imaging (DWI) sequence is often used in high-field rodent brain MRI due to its immunity to these artefacts. To accurately determine diffusion-tensor or fibre-orientation distribution, high angular resolution diffusion imaging (HARDI) with strong diffusion weighting (b >3000 s/mm2) and at least 30 diffusion-encoding directions are required. However, this results in long image acquisition times unsuitable for live animal imaging. In this study, we describe the optimization of HARDI acquisition parameters at 16.4T using a Stejskal-Tanner sequence with echo-planar imaging (EPI) readout. EPI segmentation and partial Fourier encoding acceleration were applied to reduce the echo time (TE), thereby minimizing signal decay and distortion artefacts while maintaining a reasonably short acquisition time. The final HARDI acquisition protocol was achieved with the following parameters: 4 shot EPI, b = 3000 s/mm2, 64 diffusion-encoding directions, 125×150 μm2 in-plane resolution, 0.6 mm slice thickness, and 2h acquisition time. This protocol was used to image a cohort of adult C57BL/6 male mice, whereby the quality of the acquired data was assessed and diffusion tensor imaging (DTI) derived parameters were measured. High-quality images with high spatial and angular resolution, low distortion and low variability in DTI-derived parameters were obtained, indicating that EPI-DWI is feasible at 16.4T to study animal models of white matter (WM) diseases.