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.