We performed a systematic immunohistochemical study on 378 brain tumors using 37 antibodies and tissue microarray (TMA) technology. The aim of this study was to find new diagnostic biomarkers using antibodies established in our laboratory. Our TMA consisted of a grid of 1.5-mm cores that were extracted from individual donor blocks. Staining for each antibody was scored using a three-point system. We used hierarchical clustering analysis to interpret these data, which resulted in separation of all the brain tumors into seven groups. Although there were some exceptions, cases with the same histological diagnosis were generally grouped together. We then carried out statistical analyses to find the most useful antibodies for grouping of brain tumors. Ten antibodies [glial fibrillary acidic protein (GFAP), Olig2, vimentin, epithelial membrane antigen (EMA), cytokeratin (AE1/AE3), alpha-internexin, nestin, pinealocytes PP5, aquaporin-4 (AQP4) M13d and AQP4M13e] discriminated between astrocytomas and oligodendroglial tumors. Six antibodies [EMA, AE1/AE3, TUJ1, nestin, neurofilament protein-MH (NF-MH) and perivascular cells GP-1] showed significant differences between high-grade and low-grade gliomas. Our data have revealed new antibodies with potential diagnostic utility (Olig2, PP5, GP-1) and demonstrate that TMA technology is highly useful for evaluating newly established antibodies in brain-tumor research.