Abstract: Glucocorticoids (GCs) are stress hormones found in the hypothalamus-pituitary-adrenal (HPA) axis of vertebrates, which exert their action by binding to and activating the intracellular glucocorticoid receptor (GR). Amphiprion clarkii is a hermaphrodite fish, whose sex determination and gonadal development are affected by the social status of individuals. In the present study, we used a novel RNAscope in situ hybridization technique and investigated the distribution and function ofthe glucocorticoid receptor (gr) gene in brain regions from neurotomy. The coronal planes of brain of A. clarkii showed that the gr1 and gr2 mRNA positive signals were widely distributed in the telencephalon, diencephalon, midbrain and cerebellum. In particular, the abundant gr1 and gr2 genes signal were detected in subdivision 3 of the medial dorsal telencephalic area (Dm3), subdivision 2 of the medial dorsal telencephalic area (Dm2), lateral posterior part of the dorsal telencephalic area (DIp), posterior portion of the dorsal telencephalon (Dp), postcommissural part of the ventral telencephalon (Vp) of the telencephalon, parvocellular part of the parvocellular preoptic nucleus (NPOpc), anteroventral part of the parvocellular preoptic nucleus (NPOav), gigantocellular part of the magnocellular preoptic nucleus (PMgc), anterior periventricular nucleus (NAPv) of the preoptic area, dorsal part of the nucleus of the lateral recess (NRLd), ventral part of the lateral tuberal nucleus (NLTv), nucleus of the posterior recess (NRP), inferior part of the lateral tuberal nucleus (NLTI), lateral part of the diffuse nucleus of the inferior lobe (NDLIl) of the hypothalamus, and optic tectum (OT) of the midbrain. However, the gr2 gene expression was higher in abundance than gr1 in habenular nucleus (Hb), periventricular nucleus of the posterior tuberculum (TPp), posterior part of glomerular nucleus (NGp), commissural preglomerural nucleus (NPGc), nucleus of the medial longitudinal fasciculus (nMLF), posterior tuberal nucleus (NPT) of the diencephalon, tegmentum (TEG) of the midbrain, and corpus of the cerebellum (CCe). The present study suggests that GR genes in the brain of A. clarkii may be involved in the physiological activities of emotional regulation, memory, spatial learning, stress regulation, vision, motion balance and reproductive regulation. The result provides theoretical evidence for the study of the regulation of glucocorticoids and their receptors in the central nervous system.