Objective <p>Stereotactic biopsy (STB) of brainstem lesions remains associated with neurological morbidity due to the dense eloquent tracts. Conventional intraoperative mapping cannot be directly applied in the stereotactic setting. We evaluated the feasibility, safety, and clinical relevance of intraoperative tract mapping through the inner cannula of the biopsy needle during brainstem STB.</p> Methods <p>In a prospective consecutive series, 16 patients underwent frame-based STB of intrinsic brainstem lesions. Mapping of corticospinal, corticonuclear, and medial lemniscus pathways was performed through a dual epidural D-wave electrode inserted into the biopsy cannula at the planned target depth. Sampling proceeded only at sites without tract responses; needle orientation was adjusted (first by rotation, then by depth correction) when responses were detected. Neurological status was assessed preoperatively and at discharge.</p> Results <p>Tract responses were identified in 9 patients (56%). Corticospinal responses were recorded in 5 (31%), medial lemniscus responses in 6 (38%), and corticonuclear responses in 2 (12%). Mapping indicated proximity of the biopsy target to eloquent pathways in multiple cases, prompting needle orientation adjustments. Neurological worsening occurred in 4 patients (25%), including 2 severe deficits. The most severe deficit occurred in a medullary lesion where persistent corticobulbar responses indicated direct tract involvement. No permanent deterioration occurred in patients without tract responses and without CT-proven complications.</p> Conclusions <p>Intraoperative tract mapping through the biopsy cannula during brainstem STB is feasible and compatible with awake procedures. Detecting tract proximity provides functional information at the sampling site and may reduce the risk of neurological injury.</p>