Functional MRI (fMRI) is widely used as a non-invasive method for the evaluation of pre-operation motor function. However, patients with cortical function impairment, such as those with hemiparesis, can rarely achieve hand clenching, a typical fMRI task for central sulcus identification, and the method is also of limited use in uncooperative children. Thus, it is important to develop a new method for identifying primary motor areas (PMA) in such individuals. This study used corticospinal tractography to identify the PMA in 20 patients with deep-seated brain tumor. Two regions of interest were set within the brainstem for corticospinal tract (CST) fiber tracking: one at the level of the pons and the other at the level of the cerebral peduncle. The CST fiber tracking results and fMRI activation signals were merged with three-dimensional anatomic MRI findings. The consistency of identifying the PMA by CST and fMRI was analyzed. fMRI activation signals were distributed mainly in the contralateral central sulcus around the omega-shaped hand knob. The CST consistently propagated from the pons and cerebral peduncle to the suspected PMA location. There was a good correlation between CST fiber tracking results and fMRI activation signals in terms of their abilities to identify the PMA. The differences between fMRI and CST fiber tracking findings may result from our functional task, which consisted only of hand movements. Our results indicate that diffusion tensor imaging is a useful brain mapping technique for identifying the PMA in paralyzed patients and uncooperative children.