High-grade gliomas possess internal pathological heterogeneity. Selective sampling of different tumor regions can help in the study of this heterogeneity. In this report, we have described the use of a novel navigation and optical system for the selective regional sampling of a high-grade glioma lesion. A 45-year-old gentleman presented to us with complaints of intermittent frontal headaches for past eight months. On examination, he had subtle pyramidal weakness in left upper and lower extremities. Magnetic resonance imaging (MRI) showed a large contrast-enhancing, space-occupying lesion in the right frontal lobe causing perilesional edema and midline shift. We marked four different regions on the preoperative MRI using apparent diffusion coefficient (ADC) mapping and contrast enhancement pattern in four different combinations using presurgical planning software (BrightMatter™ Plan) (Synaptive Medical, Inc., Toronto, Canada). These pre-identified areas were exported into BrightMatter™ Servo (Synaptive Medical, Toronto, Canada), an integrated robotic video microscope with a neuronavigation system where these areas were selectively sampled and sent for analysis. The BrightMatter™ Servo not only helped us to the target areas but also helped to identify a safe trajectory, respecting white matter tracts. Histopathology showed a neoplastic lesion composed of mononuclear round cells with the perinuclear halo in a fibrillary stroma with admixed mini-gemistocytes consistent with the diagnosis of a Grade 3 anaplastic astrocytoma. A selective regional sampling of the gliomas can be reliably performed using BrightMatter™ technologies to study the pathological heterogeneity of these lesions.