Kisspeptin and G protein-coupled receptor 54 (GPR54) are now acknowledged to play essential roles in the neural regulation of fertility. Using a transgenic Gpr54 LacZ knock-in mouse model, this study aimed to provide 1) a detailed map of cells expressing Gpr54 in the mouse brain and 2) an analysis of Gpr54 expression in GnRH neurons across postnatal development. The highest density of Gpr54-expressing cells in the mouse central nervous system was found in the dentate gyrus of the hippocampus beginning on postnatal d 6 (P6). Abundant Gpr54 expression was also noted in the septum, rostral preoptic area (rPOA), anteroventral nucleus of the thalamus, posterior hypothalamus, periaqueductal grey, supramammillary and pontine nuclei, and dorsal cochlear nucleus. No Gpr54 expression was detected in the arcuate and rostral periventricular nuclei of the hypothalamus. Dual-labeling experiments showed that essentially all Gpr54-expressing cells in the rPOA were GnRH neurons. Analyses of mice at birth, P1, P5, P20, and P30 and as adults revealed a gradual increase in the percentage of GnRH neurons expressing Gpr54 from approximately 40% at birth through to approximately 70% from P20 onward. Whereas GnRH neurons located in the septum displayed a consistent increase across this time, GnRH neurons in the rPOA showed a sharp reduction in Gpr54 expression after birth (to approximately 10% at P5) before increasing to the 70% expression levels by P20. Together these findings provide an anatomical basis for the exploration of Gpr54 actions outside the reproductive axis and reveal a complex temporal and spatial pattern of Gpr54 gene expression in developing GnRH neurons.