Thalamic gliomas represent a great challenge for neurosurgeons because of the high surgical risk of damaging the surrounding anatomy. Preoperative planning may considerably help the surgeon find the most ideal operative trajectory, avoiding thalamic nuclei and important white matter pathways adjacent to the tumor tissue. Thalamic segmentation is a promising imaging tool based on diffusion tensor magnetic resonance imaging. It provides the possibility to predict the relationship of the tumor to thalamic nuclei. To propose a new tool in thalamic glioma surgery that may help to differentiate between normal thalamus and tumor tissue, making preoperative planning possible and facilitating the choice of the optimal surgical approach and trajectory for neuronavigation-assisted surgery. Four patients with thalamic gliomas preoperatively underwent conventional and diffusion-weighted magnetic resonance imaging conducted on 1.5 T. Subsequently, probabilistic tractography and thalamic segmentation were performed with the FSL Software as preoperative planning. We also present a case when thalamic segmentation was applied retrospectively using preoperative images. All patients went through neuronavigation-assisted surgery (1 partial, 4 subtotal resections). Surgery performed based on the output of thalamic segmentation caused no deterioration in the neurological symptoms of our patients. Indeed, we noticed improvement in the neurological condition in 3 cases; furthermore, in 2 patients, a concern-free state was achieved. We suggest that thalamic segmentation may be applied successfully and routinely in the surgical treatment of thalamic gliomas.