Immunotherapeutic targeting of the PD-1/PD-L1 axis has been widely implemented for treatment of several cancer types but shown disappointing results in glioblastomas (GBMs), potentially due to compensatory mechanisms of other expressed immune checkpoints. Galectin-9 is an immune-checkpoint protein that facilitates T-cell exhaustion and apoptosis and could be a potential target for immune-checkpoint inhibition. A total of 163 GBMs IDH wildtype were immunostained with anti-Galectin-9 and PD-L1 antibodies. Software-based quantitation of immunostainings was performed and co-expression was investigated using double immunofluorescence. Both Galectin-9 and PD-L1 protein expression were found in all 163 tumors and showed a significant positive correlation (p = 0.0017). Galectin-9 expression varied from 0.01% to 32% (mean = 6.61%), while PD-L1 membrane expression ranged from 0.003% to 0.14% (mean = 0.048%) of total tumor area. Expression of Galectin-9 and PD-L1 was found on both microglia/macrophages and tumor cells, and colocalization of both markers was found in 88.3% of tumors. In multivariate analysis, neither Galectin-9 (HR = 0.99), PD-L1 (HR = 1.05), nor their combinations showed prognostic value. Galectin-9 and PD-L1 were expressed in all investigated GBMs and the majority of patients had co-expression, which may provide rationale for multi-targeted immune checkpoint inhibition.