The bilateral symmetry of flounder larvae changes through the process of morphogenesis to produce external asymmetry at metamorphosis. The process is characterized by the lateral migration of one eye and pigmentation at the ocular side. Migration of the left or right eye to produce either dextral or sinistral forms, respectively, is usually fixed within a species. Here we propose a mechanism for the mediation of lateralization by the nodal-lefty-pitx2 (NLP) pathway in flounders, in which pitx2, the final left-right determinant of the NLP pathway, is re-expressed in the left habenula at pre-metamorphosis. After the initiation of left-sided pitx2 re-expression, the eye commences migration, when the habenulae shift their position on the ventral diencephalon rightwards in sinistral flounder (Paralichthys olivaceus) and leftwards in dextral flounder (Verasper variegatus). In addition, the right habenula increases in size relative to the left habenula in both species. Loss of pitx2 re-expression induces randomization of eye-sidedness, manifesting as normal, reversed or bilateral symmetry, with laterality of the structural asymmetry of habenulae being entirely inverted in reversed flounders compared with normal ones. Thus, flounder pitx2 appears to be re-expressed in the left habenula at metamorphosis to direct eye-sidedness by lateralizing the morphological asymmetry of the habenulae.