The development of cannabinoid receptor type 2 (CB2R) PET radioligands has been intensively explored due to the pronounced CB2R upregulation in various pathological conditions, such as neuroinflammation and cancer. Herein we report on the enantioselective synthesis of a series of highly affine fluorinated indole-2-carboxamide ligands targeting the CB2R in the brain. Compound RM365 was selected for PET radiotracer development due to its high CB2R affinity (Ki = 2.1 nM) and pronounced selectivity over CB1R (factor > 300). A fully automated copper-mediated radiofluorination of [18F]RM365 was established starting from the corresponding aryl boronic acid pinacol ester precursor. Preliminary in vitro evaluation of [18F]RM365 indicated species differences in the binding to CB2R (KD of 2.32 nM for the human CB2R vs. KD > 10000 nM for the rat CB2R). Metabolism studies in mice revealed high stability of [18F]RM365 with fractions of parent compound of > 90% in the brain and > 54% in the plasma at 30 min p.i. PET imaging in a rat model of local hCB2R(D80N) overexpression in the brain demonstrate the ability of [18F]RM365 to reach and selectively label the intracranial expressed hCB2R(D80N) with high signal-to-background ratio. Thus, [18F]RM365 is a very promising PET radioligand for the imaging of upregulated hCB2R expression under pathological conditions with high potential towards clinical application in humans.