Prostate-specific membrane antigen (PSMA) targeting radiopharmaceuticals have been successfully used for the diagnosis and therapy of prostate cancer. Most of the PSMA molecules for the diagnosis and treatment are based on the peptidomimetic glutamate-urea-lysine (Glu-CO-Lys) pharmacophore connected to various linker groups. Optimization of the available agents is desirable to improve tumor uptake and reduce uptake in non-target organs. This can be achieved, for instance, via linker modifications and/or multivalent approaches. In this study, we synthesized several new Glu-CO-Lys-based PSMA ligands, each connected to different linkers to explore the role of these linkers on cell binding and tumor targeting potential. Additionally, a bivalent (bis) PSMA ligand, containing two PSMA targeting motifs (Glu-CO-Lys) in the same structure, was synthesized by conventional Fmoc-based solid-phase synthe-sis. DOTA- or Aoa-coupled PSMA conjugates showed high radiolabeling efficiency (≥ 90%) with [68Ga] and [18F] and resulted in the formation of one major radiolabeled product. Also, a high stability of the PSMA conjugates was found in human plasma. The [68Ga/18F]-labeled PSMA ligands exhibited the nanomolar affinity (<95 nM) specific to the PSMA-positive LNCaP tumor cell line. In the PSMA-positive tumor xenograft model, the radiolabeled PSMA ligands exhibited rapid clearance from the blood and excretion primarily via the renal system. Biodistribution and imaging studies revealed high accumulation of bis-PSMA ligand in LNCaP tumor xenografts. These render that bis-PSMA may be a promising ligand for diagnostic imaging of PSMA-positive pros-tate cancer.