Diffuse infiltrating gliomas are the most common tumors of the central nervous system (CNS), naturally progressing from a lower-grade to a higher-grade malignancy. Several genetic alterations have been correlated with astrocytic tumors; however, a number of as yet unknown genes may also be involved. Therefore, we set out to search for genes that are differentially expressed in anaplastic astrocytoma and normal CNS tissue by applying a PCR-based subtractive hybridization approach, namely, representational difference analysis (RDA). The results of DNA sequencing of a sample (96 cDNA clones) from the subtracted library allowed the identification of 18 different genes, some of which were represented by several cDNA clones, coding for the Np95, LMO1, FCGBP, DSCAM, and taxilin proteins. Quantitative real-time PCR analysis for five of these genes was performed using samples of astrocytic tumors of different grades, confirming their higher expression when compared to non-tumoral CNS tissue. Identification of differentially expressed genes present in gliomas but not in normal CNS tissue is important not only to better understand the molecular basis of these cancers, but also to generate diagnostic DNA chips, which may be useful in future therapeutic intervention.