Flatfish metamorphosis is characterized by extensive tissue remodeling, associated with a transition from pelagic to benthic lifestyle, being the migration of one eye the most dramatic change. Epigenetic mechanisms exert a pivotal role in developmental programs. This study investigates the DNA methylation profiles of migrating and non-migrating eyes using reduced-representation bisulfite sequencing (RRBS) during three developmental stages of turbot: pre-metamorphosis, climax and post-metamorphosis. Over 31% of all identified regions were hypermethylated during climax stage in both eyes, coinciding with elevated expression of the dnmt3a gene, responsible for de novo methylation. Additionally, transcription factors crucial for retinal ganglion cells (RGCs) development, including the eomesa and tbr1b, exhibited differential methylation and expression between the migrating and non-migrating eye during the climax phase. These findings underscore the significance of DNA methylation in the intricate remodeling of the visual system during turbot metamorphosis, particularly regarding RGC-mediated ocular migration and the transmission of visual signals.