In this study, linear poly(ethylene glycol) (PEG) and novel linear-hyperbranched, amphiphilic polyglycerol (hbPG) polymers with cholesterol (Ch) as a lipid anchor moiety were radiolabeled with fluorine-18 via copper-catalyzed click chemistry. In vivo investigations via positron emission tomography (PET) and ex vivo biodistribution in mice were conducted. A systematic comparison to the liposomal formulations with and without the polymers with respect to their initial pharmacokinetic properties during the first hour was carried out, revealing remarkable differences. Additionally, cholesterol was directly labeled with fluorine-18 and examined likewise. Both polymers, Ch-PEG27-CH2-triazole-TEG-(18)F and Ch-PEG30-hbPG24-CH2-triazole-TEG-(18)F (TEG: triethylene glycol), showed rapid renal excretion, whereas the (18)F-cholesten displayed retention in lung, liver, and spleen. Liposomes containing Ch-PEG27-CH2-triazole-TEG-(18)F revealed a hydrodynamic radius of 46 nm, liposomal Ch-PEG30-hbPG24-CH2-triazole-TEG-(18)F showed a radius of 84 nm and conventional liposomes with (18)F-cholesten 204 nm, respectively. The results revealed fast uptake of the conventional liposomes by liver, spleen, and lung. Most importantly, the novel hbPG-polymer stabilized liposomes showed similar behavior to the PEG-shielded vesicles. Thus, an advantage of multifunctionality is achieved with retained pharmacokinetic properties. The approach expands the scope of polymer tracking in vivo and liposome tracing in mice via PET.