In ex-vivo high-resolution diffusion magnetic resonance studies， the conventional diffusion-weighted spin-echo pulse （DWI-SE） sequence is difficult to satisfy the large sample requirement due to the long scan time. Multi-shot diffusion-weighted echo-planar imaging （MS-DWI-EPI） sequence， which combines echo-planar imaging （EPI） readout and k-space segmented acquisition， not only significantly improves the scanning efficiency， but also effectively reduces the common image aberration and distortion problems of single-shot EPI. However， the microstructure resolution ability of MS-DWI-EPI in ex-vivo samples still lacks systematic validation. In this study， we perform high-resolution diffusion imaging of ex-vivo mouse brains using 3D DWI-SE sequence and 3D MS-DWI-EPI sequence， and evaluate the differences between these two sequences in signal-to-noise ratio， diffusion tensor imaging （DTI） parameter estimation， and tractography performance. Experimental results show that the scanning time of the MS-DWI-EPI sequence is nearly 50% shorter while the signal-to-noise ratio of its raw b0 images is about three times higher than the DWI-SE for the same spatial and angular resolution of acquisition. In critical anatomical regions such as the corpus callosum and hippocampus， MS-DWI-EPI not only enhances the structural contrast of DTI images， but also improves the tractography. The sequence achieves a good balance between imaging efficiency and quality， providing a more efficient diffusion-weighted imaging protocol for high-throughput microstructural studies.