There are three groups of gonadotropin-releasing hormone (GnRH) neurons in the teleost brain. Midbrain GnRH neurons in the dwarf gourami send axons to various areas of the central nervous system. However, it is not clear whether midbrain GnRH neurons form a cell cluster separate from the nucleus of the medial longitudinal fasciculus (nMLF), which has been reported to project to the spinal cord. Thus, we performed a double labeling study. GnRH neurons were immunostained but were very faintly labeled with biocytin injected into the spinal cord. In contrast, nMLF neurons were strongly labeled with biocytin but were GnRH-immunonegative. GnRH neurons are distributed at almost the same rostrocaudal levels as nMLF neurons, but they constitute a separate cell group dorsocaudal to nMLF neurons.