Identification of molecular genetic alterations has become an important part of diagnosis and care of patients with brain tumors. Comparisons of immunohistochemistry (IHC) with DNA sequencing techniques have suggested that IHC is useful for identifying surrogates of mutations in gliomas; however, studies of the efficacy are relatively few. Our aim was to compare IHC in our neuropathology laboratory with a commercially available next-generation sequencing (NGS) platform, Tempus xT. We studied 212 immunohistochemically stained sections of gliomas to identify mutations of isocitrate dehydrogenase (IDH), p53, BRAF, the α-thalassemia/mental retardation syndrome X-linked protein (ATRX), and histone H3. Tempus xT NGS confirmed the IHC diagnosis of IDH1/R132H in 102 of 102 patients (100%), BRAF/V600E in 14 of 14 (100%) patients and H3/K27M in 10 of 10 (100%) patients. For p53, NGS confirmed the IHC diagnosis of mutation in 47 of 53 (87%) patients. For 6 patients, IHC was interpreted as wild-type while NGS indicated a mutation. NGS confirmed the IHC diagnosis of ATRX mutation in 29 of 31 (94%) patients. In 1 patient, IHC predicted a mutation that was not confirmed by NGS, and in another, IHC predicted wild-type, but NGS showed mutant. In 2 other patients, IHC diagnosis of ATRX mutation was equivocal; 1 was mutant and 1 was wild-type by NGS. Our single-center study suggests that IHC for IDH1/R132H, BRAF/V600E, and H3/K27M is highly reliable and may be used confidently in clinical practice. IHC for p53 and ATRX mutations is often reliable but possibly problematic, and genetic studies may be necessary to determine astrocytic or oligodendroglial differentiation.