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Abstract Cell lines have become an integral resource and tool for conducting biological experiments ever since the Hela cell line was first developed (Scherer et al. in J Exp Med 97:695–710, 1953). They not only allow detailed investigation of molecular pathways but are faster and more cost-effective than most in vivo approaches. The last decade saw many emerging model systems strengthening basic science research. However, lack of genetic and molecular tools in these newer systems pose many obstacles.Astyanax mexicanusis proving to be an interesting new model system for understanding metabolic adaptation. To further enhance the utility of this system, we developed liver-derived cell lines from both surface-dwelling and cave-dwelling morphotypes. In this study, we provide detailed methodology of the derivation process along with comprehensive biochemical and molecular characterization of the cell lines, which reflect key metabolic traits of cavefish adaptation. We anticipate these cell lines to become a useful resource for theAstyanaxcommunity as well as researchers investigating fish biology, comparative physiology, and metabolism.