Astrocytomas, oligodendrogliomas, and oligoastrocytomas, collectively referred to as diffuse gliomas, are the most common primary brain tumors. These tumors are classified by histologic similarity to differentiated astrocytes and oligodendrocytes, but this approach has major limitations in guiding modern treatment and research. Lineage markers represent a potentially useful adjunct to morphologic classification. The murine bHLH transcription factors Olig1 and Olig2 are expressed in neural progenitors and oligodendroglia and are essential for oligodendrocyte development. High OLIG expression alone has been proposed to distinguish oligodendrogliomas from astrocytomas, so we critically evaluated OLIG2 as a marker by immunohistochemical and oligonucleotide microarray analysis. OLIG2 protein is faithfully restricted to normal oligodendroglia and their progenitors in human brain. Immunohistochemical analysis of 180 primary, metastatic, and non-neural human tumors shows OLIG2 is highly expressed in all diffuse gliomas. Immunohistochemistry and microarray analyses demonstrate higher OLIG2 in anaplastic oligodendrogliomas versus glioblastomas, which are heterogeneous with respect to OLIG2 levels. OLIG2 protein expression is present but inconsistent and generally lower in most other brain tumors and is absent in non-neuroectodermal tumors. Overall, OLIG2 is a useful marker of diffuse gliomas as a class. However, expression heterogeneity of OLIG2 in astrocytomas precludes immunohistochemical classification of individual gliomas by OLIG2 alone.