The habenula is a conserved structure in the brain of vertebrates. With the aim of further understanding of the evolution of the habenular system in vertebrates, we studied the afferent and efferent connections of the habenula of the rainbow trout. Experiments included application of the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) into the habenula, telencephalon, pineal organ, posterior tubercle, and interpeduncular nucleus (IPN). The results obtained reveal a consistent pattern of habenular connections. Most afferents originate from three nuclei, one extending from the preoptic region to the rostral thalamus (the entopeduncular nucleus), the second located in the region of the hypothalamus-posterior tubercle and consisting of large bipolar cells (tuberculohabenular nucleus), and the third in the preoptic region (preoptic nucleus). A few large neurons of the locus coeruleus appeared to be labeled in some cases. The trout habenula also receives pineal and parapineal projections. Small labeled glial cells were observed in the thalamus around the fasciculus retroflexus and, sometimes, around the IPN. The most conspicuous efferents coursed in the fasciculus retroflexus to the IPN, the isthmal raphe, and the central gray. The existence of olfactohabenular or habenulotelencephalic projections is discussed.