Multiple forms of gonadotropin-releasing hormone (GnRH) are found in different vertebrates. In this study, we have cloned cDNA encoding the full-length gnrh3 and gnrh2 from zebrafish brain and characterized their structure and expression patterns. We performed phylogenetic analysis and compared conserved syntenies in the region surrounding the GnRH genes from human, chicken, pufferfish, and zebrafish genores. The gnrh3 and gnrh2 genes were mapped to LG17 and LG21, respectively. The zebrafish genome appears to lack an ortholog to human GNRH1, and the human genome appears to lack an ortholog of gnrh3. Expression of gnrh3 began in the olfactory pit at 24-26 h postfertilization and expanded to the olfactory bulb during early larval stage. Expression of gnrh2 is always in the midbrain. In addition, GnRH is also expressed in boundary cells surrounding seminiferous cysts of the testis. Thus, this detailed phylogenetic, chromosomal comparison, and expression study defines the identity and the evolutionary relationship of two zebrafish gnrh genes. We propose a model describing the evolution of gnrh genes involving ancestral duplication of the genes followed by selective loss of one gene in some teleosts.