Diffuse gliomas are aggressive brain tumors that respond poorly to immunotherapy including immune checkpoint inhibition. This resistance may arise from an immunocompromised microenvironment and deficient immune recognition of tumor cells because of low mutational burden. The most prominent genetic alterations in diffuse glioma are mutations in the isocitrate dehydrogenase (IDH) genes that generate the immunosuppressive oncometabolite d-2-hydroxyglutarate. Our objective was to explore the association between IDH mutation and presence of cells expressing the immune checkpoint proteins galectin-9 and/or T cell immunoglobulin and mucin-domain containing-3 (TIM-3). Astrocytic gliomas of World Health Organization (WHO) grades III or IV (36 IDH-mutant and 36 IDH-wild-type) from 72 patients were included in this study. A novel multiplex chromogenic immunohistochemistry panel was applied using antibodies against galectin-9, TIM-3, and the oligodendrocyte transcription factor 2 (OLIG2). Validation studies were performed using data from The Cancer Genome Atlas (TCGA) project. IDH mutation was associated with decreased levels of TIM-3⁺ cells (p < 0.05). No significant association was found between galectin-9 and IDH status (p = 0.10). Most TIM-3⁺ and galectin-9⁺ cells resembled microglia/macrophages, and very few TIM-3⁺ and/or galectin-9⁺ cells co-expressed OLIG2. The percentage of TIM-3⁺ T cells was generally low, however, IDH-mutant tumors contained significantly fewer TIM-3⁺ T cells (p < 0.01) and had a lower interaction rate between TIM-3⁺ T cells and galectin-9⁺ microglia/macrophages (p < 0.05). TCGA data confirmed lower TIM-3 mRNA expression in IDH-mutant compared to IDH-wild-type astrocytic gliomas (p = 0.013). Our results show that IDH mutation is associated with diminished levels of TIM-3⁺ cells and fewer interactions between TIM-3⁺ T cells and galectin-9⁺ microglia/macrophages, suggesting reduced activity of the galectin-9/TIM-3 immune checkpoint pathway in IDH-mutant astrocytic gliomas.