Over the last years, distinct genetic lesions have been associated with individual tumor entities. Stereotactic biopsy has become an essential diagnostic tool in surgical neuro-oncology. In order to evaluate the potential of molecular analyses in stereotactic biopsies, we examined a series of 156 human brain tumors from patients undergoing stereotactic biopsy for molecular alterations typically seen in astrocytic gliomas and compared those results with a control group of 268 astrocytic tumors obtained at open surgery. Stereotactic biopsies of astrocytomas with borderline histopathological features between the WHO grades II and III showed a higher rate of allelic losses on chromosome 10 than those of the WHO grade II from open surgery (p = 0.011). Stereotactic biopsies of astrocytomas with borderline histopathological features between the WHO grades III and IV showed a higher rate of allelic losses on chromosome 10 than those of the WHO grade III from open surgery (p = 0.013). This indicates that stereotactic biopsies with features intermediate between grades are likely to correspond to the higher malignancy grade. Our data demonstrate that molecular genetic approaches can be successfully applied to stereotactic glioma biopsies. The difference in the distribution of malignancy associated genetic alterations between a stereotactic and openly resected group of gliomas indicates that histopathology may underestimate the malignant potential in some stereotactic specimens. We propose to further evaluate the molecular analysis of stereotactic glioma biopsies as a useful adjunct to standard histopathological procedures.