Introduction: The endocannabinoid system is involved in several diseases such as addictive disorders, schizophrenia, post-traumatic stress disorder, and eating disorders. As often mice are used as the preferred animal model in translational research, in particular when using genetically modified mice, this study aimed to provide a systematic analysis of in vivo cannabinoid type 1 (CB1) receptor ligand-binding capacity using positron emission tomography (PET) using the ligand [18F]MK-9470. We then compared the PET results with literature data from immunohistochemistry (IHC) to review the consistency between ex vivo protein expression and in vivo ligand binding.Methods: Six male C57BL/6J (6–9 weeks) mice were examined with the CB1 receptor ligand [18F]MK-9470 and small animal PET. Different brain regions were evaluated using the parameter %ID/ml. The PET results of the [18F]MK-9470 accumulation in the mouse brain were compared with immunohistochemical literature data.Results: The ligand [18F]MK-9470 was taken up into the mouse brain within 5 min after injection and exhibited slow kinetics. It accumulated highly in most parts of the brain. PET and IHC classifications were consistent for most parts of the telencephalon, while brain regions of the diencephalon, mesencephalon, and rhombencephalon were rated higher with PET than IHC.Conclusions: This preclinical [18F]MK-9470 study demonstrated the radioligand’s applicability for imaging the region-specific CB1 receptor availability in the healthy adult mouse brain and thus offers the potential to study CB1 receptor availability in pathological conditions.