Gallium-68 (⁶⁸Ga) is a positron-emitting isotope used for clinical PET imaging of peptide receptor expression. ⁶⁸Ga radiopharmaceuticals used in molecular PET imaging consist of disease-targeting biomolecules tethered to chelators that complex ⁶⁸Ga³⁺. Ideally, the chelator will rapidly, quantitatively and stably coordinate ⁶⁸Ga³⁺ at room temperature, near neutral pH and low chelator concentration, allowing for simple routine radiopharmaceutical formulation. Identification of chelators that fulfil these requirements will facilitate development of kit-based ⁶⁸Ga radiopharmaceuticals. Herein the reaction of a range of widely used macrocyclic and acyclic chelators with ⁶⁸Ga³⁺ is reported. Radiochemical yields have been measured under conditions of varying chelator concentrations, pH (3.5 and 6.5) and temperature (25 and 90 °C). These chelators are: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane macrocycles substituted with phosphonic (NOTP) and phosphinic (TRAP) groups at the amine, bis(2-hydroxybenzyl)ethylenediaminediacetic acid (HBED), a tris(hydroxypyridinone) containing three 1,6-dimethyl-3-hydroxypyridin-4-one groups (THP) and the hexadentate tris(hydroxamate) siderophore desferrioxamine-B (DFO). Competition studies have also been undertaken to assess relative complexation efficiencies of each chelator for ⁶⁸Ga³⁺ under different pH and temperature conditions. Performing radiolabelling reactions at pH 6.5, 25 °C and 5–50 μM chelator concentration resulted in near quantitative radiochemical yields for all chelators, except DOTA. Radiochemical yields either decreased or were not substantially improved when the reactions were undertaken at lower pH or at higher temperature, except in the case of DOTA. THP and DFO were the most effective ⁶⁸Ga³⁺ chelators at near-neutral pH and 25 °C, rapidly providing near-quantitative radiochemical yields at very low chelator concentrations. NOTP and HBED were only slightly less effective under these conditions. In competition studies with all other chelators, THP demonstrated highest reactivity for ⁶⁸Ga³⁺ complexation under all conditions. These data point to THP possessing ideal properties for rapid, one-step kit-based syntheses of ⁶⁸Ga-biomolecules for molecular PET imaging. LC-MS and ¹H, ¹³C{¹H} and ⁷¹Ga NMR studies of HBED complexes of Ga³⁺ showed that under the analytical conditions employed in this study, multiple HBED-bound Ga complexes exist. X-ray diffraction data indicated that crystals isolated from these solutions contained octahedral [Ga(HBED)(H₂O)], with HBED coordinated in a pentadentate N₂O₃ mode, with only one phenolic group coordinated to Ga³⁺, and the remaining coordination site occupied by a water molecule.