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During extensive evolution, higher eukaryotes have acquired numerous prokaryotic genes, some of which have experienced adaptive copy number variations (CNVs) in response to changing environments. The specific contribution of ancient prokaryotic genes to the evolutionary dynamics of marine fish remains unclear. In this study, we investigated ancient prokaryotic genes in 33 freshwater and 14 non-freshwater fish species. Non-freshwater fish exhibited a higher proportion of these genes (25.84 %) compared to freshwater fish (17.66 %), though functional categories showed similarity, suggesting genetic conservation. Comprehensive CNV analysis identified 24 genes with significantly higher copy numbers in non-freshwater fish, enriched in glycerolipid metabolism, immune regulation, and catechol O -methyltransferase activity. Eight of these genes displayed increased copy numbers in marine populations compared to Asian freshwater populations. These findings indicate that CNVs in genes related to glycerolipid metabolism, immune regulation, and methyltransferase activity may play crucial roles in marine adaptation at global and regional evolutionary scales. [Display omitted] • Ancient prokaryotic genes and domains were found in 47 freshwater and marine fish species. • Sixteen genes were identified with increased copy numbers associated with marine adaptation. • Fish lineage evolution is influenced by ancient prokaryotic genes' evolution. • Marine fish colonization involves old prokaryotic and new genes with higher copies. [ABSTRACT FROM AUTHOR]