Spexin (SPX) is a highly conserved neuropeptide that is widely expressed in mammalian brain and peripheral tissue. In teleost, SPX1 is mainly expressed in the brain and ovary, and is involved in reproduction and food intake. A second form of SPX, SPX2, was recently identified in chick, Xenopus, and zebrafish. The expression pattern and roles of SPX2 are unknown. SPX (spx1) is highly expressed in the vertebrate brain, but its distribution, circuits, and interactions with its putative receptor are unknown. Here, we observed expression of spx1 in the midbrain and hindbrain, and spx2 in the hypothalamic preoptic area using in situ RNA hybridization in zebrafish. Analysis of transgenic reporter zebrafish revealed that hindbrain SPX1 neurons are PAX2+ inhibitory interneurons and project to the spinal cord, where they interact with galanin receptor 2b (GALR2b) neurons, suggesting that hindbrain SPX1 neurons are reticulospinal neurons. spx1 mRNA and SPX1 reporter expression were observed in dorsal habenula (dHb). SPX1 neurons in the dHb project to the interpeduncular nucleus (IPN), where GALR2a and GALR2b expression was also observed, suggesting that habenula SPX1 neurons may interact with GALR2a/2b in the IPN.