We used a Tg(glyt2:gfp) transgenic zebrafish expressing the green fluorescent protein (GFP) under control of the glycine transporter 2 (GLYT2) regulatory sequences to study for the first time the glycinergic neurons in the brain of an adult teleost. We also performed in situ hybridization using a GLYT2 probe and glycine immunohistochemistry. This study was combined with biocytin tract tracing from the spinal cord to reveal descending glycinergic pathways. A few groups of GFP+/GLYT2− cells were observed in the midbrain and forebrain, including numerous pinealocytes. Conversely, a small nucleus of the midbrain tegmentum was GLYT2+ but GFP−. Most of the GFP+ and GLYT2+ neurons were observed in the rhombencephalon and spinal cord, and a portion of these cells showed double GLYT2/GFP labeling. In the hindbrain, GFP/GLYT2+ populations were observed in the medial octavolateral nucleus; the secondary, magnocellular, and descending octaval nuclei; the viscerosensory lobes; and reticular populations distributed from trigeminal to vagal levels. No glycinergic cells were observed in the cerebellum. Tract tracing revealed three conspicuous pairs of GFP/GLYT2+ reticular neurons projecting to the spinal cord. In the spinal cord, GFP/GLYT2+ cells were observed in the dorsal and ventral horns. GFP+ fibers were observed from the olfactory bulbs to the spinal cord, although their density varied among regions. The Mauthner neurons received very rich GFP+ innervation, mainly around the axon cap. Comparison of the zebrafish glycinergic system with the glycinergic systems of other adult vertebrates reveals shared patterns but also divergent traits in the evolution of this system. J. Comp. Neurol. 521:389–425, 2013. © 2012 Wiley Periodicals, Inc.