Here, we used high throughput next-generation sequencing (NGS) to identify molecular and biological processes involved in the responses to environmental changes. Preliminary tests revealed that seawater (SW)-acclimated milkfish tolerated lower temperatures than the fresh water (FW)-acclimated group. Although FW- and SW-acclimated milkfish have different levels of tolerance for hypothermal stress, to date, the molecular physiological basis of this difference has not been elucidated. Here, we performed a next-generation sequence analysis of mRNAs from four groups of milkfish.We obtained 29669 unigenes with an average length of approximately 1936 base pairs. Gene ontology (GO) analysis was performed after gene annotation. A large number of genes for molecular regulation were identified through a transcriptomic comparison in a KEGG analysis. Basal metabolic pathways involved in hypothermal tolerance, such as glycolysis, fatty acid metabolism, amino acid catabolism and oxidative phosphorylation, were analyzed using PathVisio and Cytoscape software. Our results indicate that in response to hypothermal stress, genes for oxidative phosphorylation, e.g., succinate dehydrogenase, were more highly up-regulated in SW than FW fish. Moreover, SW and FWmilkfish used different strategies when exposed to hypotherma stress: SW milkfish up-regulated oxidative phosphorylation and catabolism genes to produce more energy budget, whereas FWmilkfish down-regulated genes related to basal metabolism to reduce energy loss.