The present study characterizes gonadotropin-releasing hormone (GnRH) neuronal groups that are located in several different brain regions by investigating GnRH molecular species and projection patterns in an anabantid fish, Colisa lalia. First, we examined the molecular species of GnRHs in extracts of the brain and the pituitary by reverse-phase high-performance liquid chromatography followed by radioimmunoassays. We found salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and an unfamiliar GnRH-like substance. Next, to examine the distribution of each GnRH molecule in different GnRH neuronal groups, we performed immunohistochemistry using four kinds of antisera and an antibody. Furthermore, we performed brain lesioning experiments of terminal nerve (TN) cells, the most conspicuous GnRH-immunoreactive cells in Colisa lalia. Comparisons of immunoreactive structures between TN-lesioned fish and untreated fish elucidated the projection area of each neuronal group. Three major neuronal groups were observed. TN-GnRH cells, which are located in the transitional area between the olfactory bulb and the telencephalon, showed strong sGnRH and weaker cGnRH-II immunoreactivity. TN-GnRH cells projected to wide areas of the central nervous system from the olfactory bulb to the spinal cord. The second group, located in the preoptic area, showed only sGnRH immunoreactivity and projected only to the pituitary. The third one, located in the midbrain tegmentum, exhibited strong cGnRH-II and weaker sGnRH immunoreactivity. This cell group projected mainly to brain regions posterior to the hypothalamus and the spinal cord. These different projection patterns suggest functional differentiation of each GnRH neuronal group.