Abstract Purpose CXCR4 (over)expression is found in multiple human cancer types, while expression is low or absent in healthy tissue. In glioblastoma it is associated with a poor prognosis and more extensive infiltrative phenotype. CXCR4 can be targeted by the diagnostic PET agent [⁶⁸Ga]Ga-Pentixafor and its therapeutic counterpart [¹⁷⁷Lu]Lu-Pentixather. We aimed to investigate the expression of CXCR4 in glioblastoma tissue to further examine the potential of these PET agents. Methods CXCR4 mRNA expression was examined using the R2 genomics platform. Glioblastoma tissue cores were stained for CXCR4. CXCR4 staining in tumor cells was scored. Stained tissue components (cytoplasm and/or nuclei of the tumor cells and blood vessels) were documented. Clinical characteristics and information on IDH and MGMT promoter methylation status were collected. Seven pilot patients with recurrent glioblastoma underwent [⁶⁸Ga]Ga-Pentixafor PET; residual resected tissue was stained for CXCR4. Results Two large mRNA datasets (N = 284; N = 540) were assesed. Of the 191 glioblastomas, 426 cores were analyzed using immunohistochemistry. Seventy-eight cores (23 tumors) were CXCR4 negative, while 18 cores (5 tumors) had both strong and extensive staining. The remaining 330 cores (163 tumors) showed a large inter- and intra-tumor variation for CXCR4 expression; also seen in the resected tissue of the seven pilot patients—not directly translatable to [⁶⁸Ga]Ga-Pentixafor PET results. Both mRNA and immunohistochemical analysis showed CXCR4 negative normal brain tissue and no significant correlation between CXCR4 expression and IDH or MGMT status or survival. Conclusion Using immunohistochemistry, high CXCR4 expression was found in a subset of glioblastomas as well as a large inter- and intra-tumor variation. Caution should be exercised in directly translating ex vivo CXCR4 expression to PET agent uptake. However, when high CXCR4 expression can be identified with [⁶⁸Ga]Ga-Pentixafor, these patients might be good candidates for targeted radionuclide therapy with [¹⁷⁷Lu]Lu-Pentixather in the future.