Pediatric malignant non-brainstem glioma (PMNBG) is a rare tumor that accounts for only about five percent of childhood intracranial neoplasms. DNA topoisomerase IIalpha (TIIalpha) is a novel marker of cell-cycle turnover and a target of high-risk chemotherapy in PMNBG. We have shown that TIIalpha protein expression strongly correlates with event-free and overall survival in these malignancies. The molecular mechanism causing the varying TIIalpha protein expression in PMNBG remains unknown. Utilizing a combined approach of immunocytochemistry-based morphology guidance, laser-assisted microdissection and quantitative real-time PCR, we report a low-level co-amplification of the neighboring TIIalpha and Her-2/neu gene loci on chromosome 17q11-q22 in one of seventeen examined PMNBGs. Analysis of both genes by real-time PCR in the crude tumor samples without prior tissue heterogeneity reduction via laser microdissection, resulted in loss of detection of amplification of the syngeneic Her-2/neu locus. Gene dosage assessment in a microscopically distant tumor area revealed no amplification of either gene. Our results suggest that low-level amplification of the TIIalpha gene locus may be a sporadic mechanism of increased TIIalpha protein expression in PMNBG, which can coincide with low-level amplification of Her-2/neu. The observed intratumor genetic heterogeneity for TIIalpha in PMNBGs may have an impact on the relevance of TIIalpha as a biological constituent of outcome in these neoplasms.