Purpose: This investigation is intended to obtain differentially expressed genes related to human malignant glioma using Subtractive hybridization. Materials and Methods: Subtractive hybridization is potentially faster methods for identifying differentially expressed genes associated with a particular disease state. We identified 7 over-expressed genes which were not homologous to any of the known genes in the Genbank™ database. Results: Using semi-quantitative reverse transription-polymerase chain reaction (RT-PCR), the mRNA expression levels of these 7 genes were higher in human glioblastomas tissue than in non-tumor brain tissue. In order to learn more about the expression profile of these genes, RT-PCR was performed using various commercially available human carcinoma cell lines. Some of these new genes were over-expressed in human glioma cell line, but not the expressed in other human cancer cell line. Conclusion: Theses cloned new genes may play a role in brain tumorigenesis. Further studies including verification of oncogene, cancer protein, and glioblastoma induction in animal model are needed.