Adult fish produce new cells throughout their central nervous system during the course of their lives. Furthermore, they maintain a tremendous capacity to repair damaged neural tissue. Much of the focus on understanding brain repair and regeneration in fish has been directed at regions of the brainstem and forebrain however, the mesencephalon (midbrain) and diencephalon have received comparatively little attention. We sought to characterize differential gene expression in the midbrain/diencephalon in response to injury in the adult fish using RNA-seq. Using the mummichog (Fundulus heteroclitus), we administered a mechanical lesion traversing the midbrain optic tectum, tegmentum and underlying hypothalamus of the diencephalon and examined differential gene expression at an acute recovery time of 1hr post-injury. Comparisons of whole transcriptomes derived from isolated RNA of intact and injured midbrain/diencephalic tissue identified over 400 differentially expressed genes with the vast majority being upregulated. Using qPCR, we validated the upregulation of three differentially expressed genes, pim-2-like, syndecan-4-like, and cd83. Based on genes both familiar and novel regarding the adult brain response to injury, these data provide an extensive molecular profile giving insight into a range of cellular processes putatively involved in the injury response of a brain regenerative-capable vertebrate. Overall design: 10 subjects x 2 samples of midbrain/diencephalon tissue from each subject = 20 samples total: 10 Lesioned (L) hemispheres and 10 contralateral Intact (I) hemispheres