Background: Programmed cell death ligand 1 (PD-L1) plays a critical role in the tumor microenvironment and overexpression in several solid cancers has been reported. This was associated with a downregulation of the local immune response, specifically of T-cells. Immune checkpoint inhibitors showed a potential to break this localized immune paralysis, but only 30% of patients are considered responders. New diagnostic approaches are therefore needed to determine patient eligibility. Small molecule radiotracers targeting PD-L1, may serve as such diagnostic tools, addressing the heterogeneous PD-L1 expression between and within tumor lesions, thus aiding in therapy decisions. Results: Four biphenyl-based small-molecule PD-L1 ligands were synthesized using a convergent synthetic route with a linear sequence of up to eleven steps. As a chelator NODA-GA, CB-TE2A or DiAmSar was used to allow radiolabeling with copper-64 ([⁶⁴Cu]Cu-14–[⁶⁴Cu]Cu-16). In addition, a dimeric structure based on DiAmSar was synthesized ([⁶⁴Cu]Cu-17). All four radioligands exhibited high proteolytic stability (> 95%) up to 48 h post-radiolabeling. Saturation binding yielded moderate affinities toward PD-L1, ranging from 100 to 265 nM. Real-time radioligand binding provided more promising K_D values around 20 nM for [⁶⁴Cu]Cu-14 and [⁶⁴Cu]Cu-15. In vivo PET imaging in mice bearing both PC3 PD-L1 overexpressing and PD-L1-mock tumors was performed at 0–2, 4–5 and 24–25 h post injection (p.i.). This revealed considerably different pharmacokinetic profiles, depending on the substituted chelator. [⁶⁴Cu]Cu-14, substituted with NODA-GA, showed renal clearance with low liver uptake, whereas substitution with the cross-bridged cyclam chelator CB-TE2A resulted in a primarily hepatobiliary clearance. Notably, the monomeric DiAmSar radioligand [⁶⁴Cu]Cu-16 demonstrated a higher liver uptake than [⁶⁴Cu]Cu-15, but was still renally cleared as evidenced by the lack of uptake in gall bladder and intestines. The dimeric structure [⁶⁴Cu]Cu-17 showed extensive accumulation and trapping in the liver but was also cleared via the renal pathway. Of all tracer candidates and across all timepoints, [⁶⁴Cu]Cu-17 showed the highest accumulation at 24 h p.i. in the PD-L1-overexpressing tumor of all timepoints and all radiotracers, indicating drastically increased circulation time upon dimerization of two PD-L1 binding motifs. Conclusions: This study shows that chelator choice significantly influences the pharmacokinetic profile of biphenyl-based small molecule PD-L1 radioligands. The NODA-GA-conjugated radioligand [⁶⁴Cu]Cu-14 exhibited favorable renal clearance; however, the limited uptake in tumors suggests the need for structural modifications to the binding motif for future PD-L1 radiotracers.