RFamide peptides are expressed in the early stages of development in most vertebrates. Gonadotropin-inhibitory hormone (GnIH) belongs to the RFamide family, and its role in reproduction has been widely studied in adult vertebrates, ranging from fish to mammals. As only three reports evaluated GnIH during development, the aim of this study was to characterise the ontogeny of GnIH in a fish model, Cichlasoma dimerus. We detected the presence of two GnIH-immunoreactive (GnIH-ir) cell clusters with spatial and temporal differences. One cluster was observed by 3 days post-hatching (dph) in the nucleus olfacto-retinalis (NOR) and the other in the nucleus posterioris periventricularis by 14 dph. The number of GnIH-ir neurons increased in both nuclei, whereas their size increased only in the NOR from hatchling to juvenile stages. These changes occurred from the moment larvae started feeding exogenously and during development and differentiation of gonadal primordia. We showed by double-label immunofluorescence that only GnIH-ir neurons in the NOR co-expressed GnRH3 associated peptide. In addition, GnIH-ir fibre density increased in all brain regions from 5 dph. GnIH-ir fibres were also detected in the retina, optic tract and optic tectum, suggesting that GnIH acts as a neuromodulator of photoreception and the integration of different sensory modalities. Also, there were GnIH-ir fibres in the pituitary from 14 dph, which were in close association with somatotropes. Moreover, GnIH-ir fibres were observed in the saccus vasculosus from 30 dph, suggesting a potential role of GnIH in the modulation of its function. Finally, we found that gnih was expressed from 1 dph, and that the pattern of variation of its transcript levels was in accordance with that of cell number. Present results are the starting point for the study of new GnIH roles during development. This article is protected by copyright. All rights reserved.