Concurrent and/or progressive degeneration of upper and lower motor neurons (LMNs) causes neurological symptoms and dysfunctions in motor neuron diseases (MNDs) such as amyotrophic lateral sclerosis (ALS). Although brain lesions are readily detected, magnetic resonance imaging of the brainstem and cervical spinal cord lesions resulting from damage to LMNs has proven to be difficult. With the development of mouse models of MNDs, a noninvasive neuroimaging modality capable of detecting lesions resulting from axonal and neuronal injury in mouse brainstem and cervical spinal cord could improve our understanding of the underlying mechanism of MNDs and aid in the development of effective treatments. Here we present a protocol that allows the concomitant acquisition of high-quality in vivo full-diffusion tensor magnetic resonance images from the mouse brainstem and cervical spinal cord using the actively decoupled, anatomically shaped pair of coils-the surface-receive coil and the minimized volume-transmit coil. To improve the data quality, we used a custom-made nose cone to monitor respiratory motion for synchronizing data acquisition and assuring physiological stability of mice under examination. The protocol allows the acquisition of in vivo diffusion tensor imaging of the mouse brainstem and cervical spinal cord at 117 μm × 117 μm in-plane resolution with a 500-μm slice thickness in 1 h on a 4.7-T horizontal small animal imaging scanner equipped with an actively shielded gradient coil capable of pulsed gradient strengths up to 18 G cm-1 with a gradient rise time of ≤295 μs.