Glioblastoma is the most frequent and aggressive primary brain cancer in adults, and the prognosis is poor. The neurotrophic factor glial cell-derived neurotrophic factor (GDNF) and its receptors, which are involved in neuronal development, have in experimental studies been suggested to drive tumorigenic processes in glioblastoma, but the role and expression in glioblastoma in patients is under-investigated. The aim of this study was to investigate the expression of GDNF, GDNF family receptor 1-4 (GFRA1-4), and the downstream REarranged during Transfection (RET) receptor in human glioblastoma tissue by RNA in situ hybridization, immunohistochemistry, and immunofluorescence. Expression was quantified by software-based classifiers. The results showed that GDNF was expressed in approximately 10% of tumor cells. The GFRA1 receptor was widely expressed in tumor cells, often colocalizing with the astrocytic tumor cell marker glial fibrillary acidic protein (GFAP), and in a smaller fraction of tumor cells expressing the stem cell markers oligodendrocyte transcription factor 2 (OLIG2) and SRY-Box Transcription Factor 2 (SOX2). The GFRA2 receptor expression was very limited, whereas expression of GFRA3, GFRA4, and RET, respectively, was almost absent. In conclusion, GDNF and its primary receptor GFRA1 were expressed in patient glioblastoma tissue. Potential clinical value needs further investigation.