Expression of programmed cell death ligand 1 (PD-L1) within tumors is an important biomarker for guiding immune checkpoint therapies; however, immunohistochemistry-based methods of detection fail to provide a comprehensive picture of PD-L1 levels in an entire patient. To facilitate quantification of PD-L1 in the whole body, we developed a peptide-based, high-affinity PD-L1 imaging agent labeled with 18Ffluoride for positron emission tomography (PET) imaging. The parent peptide, WL12, and the nonradioactive analog of the radiotracer, 19FPy-WL12, inhibit PD-1/PD-L1 interaction at low nanomolar concentrations (half maximal inhibitory concentration IC50, 26-32 nM). The radiotracer, 18FFPy-WL12, was prepared by conjugating 2,3,5,6-tetrafluorophenyl 6-18Ffluoronicotinate (18FFPy-TFP) to WL12 and assessed for specificity in vitro in 6 cancer cell lines with varying PD-L1 expression. The uptake of the radiotracer reflected the PD-L1 expression assessed by flow cytometry. Next, we performed the in vivo evaluation of 18FFPy-WL12 in mice bearing cancer xenografts by PET imaging, ex vivo biodistribution, and blocking studies. In vivo data demonstrated a PD-L1-specific uptake of 18FFPy-WL12 in tumors that is reduced in mice receiving a blocking dose. The majority of 18FFPy-WL12 radioactivity was localized in the tumors, liver, and kidneys indicating the need for optimization of the labeling strategy to improve the in vivo pharmacokinetics of the radiotracer.