We report a molecular stereotactic biopsy technique that combines histopathologic diagnosis with small sample size-adjusted molecular genetic analysis of low-grade gliomas that are ineligible for tumor resection. Loss of heterozygosity (LOH) of 1p/19q and TP53 mutations were analyzed in 1-mm tissue samples from 42 World Health Organization grade II gliomas (30 astrocytomas, 8 oligoastrocytomas, 4 oligodendrogliomas) using polymerase chain reaction-based microsatellite and sequence analysis. Alternating histological and molecular genetic evaluation within 1-mm steps at different sites within each tumor was performed to determine reproducibility of the results and the intratumoral distribution of the biomarkers. Multiple serial biopsies (range, 2-5 per tumor) taken from distinct intratumoral areas revealed concordant molecular genetic findings and homogeneous distribution of both biomarkers throughout 41 tumors. Contamination by nonneoplastic tissue could be recognized by corresponding histological evaluation and resulted in discordant LOH findings in 1 tumor. The frequency of LOH 1p/19q and TP53 mutations was consistent with the literature; these genetic alterations were found to be mutually exclusive. There was no biopsy-related morbidity. We conclude that determination of the LOH 1p/19q and TP53 status using this molecular stereotactic biopsy technique is safe and reliable in cases of unresectable gliomas.