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INTRODUCTION: Imaging of the course of the corticospinal tract (CST) by diffusion tensor imaging (DTI) is useful for function-preserving tumour surgery especially of gliomas infiltrating the white matter. The integration of functional data such as functional MRI (fMRI) or navigated transcranial magnetic stimulation (nTMS) for the delineation of starting Regions of Interest (ROI) can improve the fibre tracking results in a multiple-ROI approach. Here, both functional localizer methods are compared for the plausibility of the resulting fibre course. METHODS: 32 patients with intracerebral tumours were allocated to the prospective study. The cortical seeding ROIs were defined by the nTMS hot-spots or by the fMRI local-activation-maximum representing the primary motor area (M1) of the hand, the foot and the tongue. The CST originating from the contralesional M1 hand area was mapped as intra-individual reference. As subcortical ROI, we used the anterior inferior pontine region (aiP). The plausibility of the fibre trajectories was assessed by a-priori defined anatomical criteria by two independent raters. As potential co-factors i.a. the franctional anisotropy threshold (FAT) was analysed. RESULTS: Both methods revealed excellent plausibility results for the contralesional hand-CST (100%). NTMS seems to be favourable to construct the hand-CST on the lesional side (NTMS/fMRI: 97%/81%) whereas no significant difference could be observed for the foot- and the tongue-CST. Plausibility rates were significantly deteriorated at low FAT values (ROC-cut-off: FAT = 0.105) for both nTMS- and fMRI-derived CST (p < 0.001). The interrater reliability was excellent (Cohen's k = 0.96). CONCLUSIONS: In summary, we found excellent plausibility rates using a deterministic multiple-ROI approach with a cortical ROI defined by functional localizer methods. In comparison, nTMS was advantageous for the hand-CST tractography whereas both localizer methods revealed adequate results for the foot- and the tongue-CST. In general, tractography results at FAT lower than 0.105 should be interpreted with particular caution.