Phosphatase and tensin homolog (PTEN) loss and epidermal growth factor receptor (EGFR) amplification are common genetic alterations in malignant gliomas. This study aimed to investigate the anatomical relationship of tumor-related PTEN and EGFR expression in 140 patients with histologically confirmed de novo glioblastoma. Preoperative magnetic resonance images were retrospectively analyzed. The lesions of each patient were segmented manually and registered to a standard brain space. Overlaying of the lesions was performed, and specific brain regions associated with PTEN loss and EGFR amplification were identified by voxel-based lesion-symptom mapping analyses. A cluster located in the right frontal lobe was found to be associated with high occurrence of PTEN loss, whereas a cluster in the right parietal lobe was demonstrated to be specifically associated with high occurrence of EGFR amplification. An overlap of the two clusters was observed at the posterior portion of the right parietal lobe. Based on voxel-based imaging analyses, our results suggest that genetic changes during the tumorigenic process may have anatomical specificity. We hope that this identified correlation between these biomarkers and the anatomical distribution of glioblastomas will help enhance our understanding of the molecular mechanisms underlying glioblastoma development and progression.