Diffuse gliomas are the most common brain tumors and include astrocytomas, oligodendrogliomas, and oligoastrocytomas. Their correct pathologic diagnosis requires the ability to distinguish astrocytic from oligodendroglial differentiation in histologic sections, a challenging feat even for the most experienced neuropathologist. Interobserver variability in the diagnosis of diffuse gliomas has been high owing to subjective diagnostic criteria, overlapping morphologic features, and variations in training and practice among pathologists. A select, albeit imperfect, group of molecular and immunohistochemical tests are available to assist in diagnosis of these lesions. Combined loss of chromosomes 1p and 19q is a genetic signature of oligodendrogliomas, whereas gains of chromosome 7 in the setting of intact 1p/19q are more typical of astrocytomas. Detection of amplified epidermal growth factor receptor favors the diagnosis of high-grade astrocytomas over anaplastic oligodendroglioma, which is especially relevant for small cell astrocytomas. Strong nuclear staining for p53 often reflects TP53 mutation and is typical of low-grade astrocytomas. The Olig family of transcription factors has not demonstrated their diagnostic usefulness. Diffuse gliomas remain a diagnostic challenge, and new markers are needed for proper classification and directed therapies.