Evidence suggest that estrogens play crucial roles in the regulation of neural development and function and the G protein-coupled estrogen receptor (GPER/GPR30) appears to be the predominant estrogen receptor in the brain. However, the distribution and functions of GPER in the developing and mature brain are not fully understood. The current study aimed to characterize the expression of GPER in the forebrain, using Gper gene reporter mice combined with fluorescent in situ hybridization (FISH/RNAscope) and immunohistochemistry (IHC). Two lines of Gper reporter mice were constructed by crossing the Gper-cre mice with Ai14(RCL-tdT)-D or R26-ZsGreen mice, which showed identical spatial distributions of the reporters in adult brain. In the forebrain, neurons, protoplasmic astrocytes, mural cells and ependymal cells of third ventricle, were found to express Gper reporters. GPER-expressing neurons were particularly enriched in the olfactory system and the salience network, including posteromedial nucleus of the cortical amygdala (PmCo), entorhinal cortex, insula cortex, prefrontal cortex and dentate gyrus of the hippocampus. RNAscope and neural tracing showed GPER-expressing cortical neurons were long-range excitatory pyramidal neurons. GPER-expressing astrocytes represented a minor population (<
10 %) of astrocytes and were found to be closely associated with neurovascular units. GPER-expressing mural cells were not labelled by the common pericyte marker PDGFRβ. In the critical period of neural development (P1-P10), GPER expression appeared to be intimately associated with neurogenesis, proliferation and migration in the olfactory system and the salience network. Collectively, the spatial and temporal pattern of GPER/GPR30 expression in the forebrain implied it might play important roles regulating the development and functions of the olfactory system, the salience network and the cerebral vessels.