This study aimed to investigate the influence of dietary supplementation with fish oil (OP), rich in 22:6n3 (DHA) and 20:5n3 (EPA); linseed oil (OL), rich in 18:3n3 (LNA); and coconut oil (OC), rich in saturated fatty acids in the lipid metabolism of Epinephelus marginatus (dusky grouper) adult female. Additionally it was performed sequencing and de novo assembly of the E. marginatus liver transcriptome by Next Generation Sequence, and sequences obtained were used as a tool to validate lipid-relevant genes. The results showed that the lipid concentration in the liver was higher in the control group (fed only with sardine) possibly due to a reduced digestibility of the incorporated fatty acids in the diet from the other experimental groups. In general the triglycerides (TG) of tissues reflected more the diet profile than phospholipids (PL), and were characterized by the high percentages of 18:3n3 in the OL group in all tissues, and DHA in the muscles of the OP group. Apparently the results for the saturated and monounsaturated fatty acids suggest a self-regulatory potential for retention and oxidation processes for this species, since in general the tissues did not directly reflect the fatty acid diet composition. The ELOVL5 expression presumably influenced in the fatty acids intermediate deposition in the animal tissue from all experimental groups, which were not seen or not representative in the experimental diets. The sequencing of the transcripts from E. marginatus liver generated 92,887 high quality contigs, and it was observed a higher number of functional annotations against zebrafish (Danio rerio) proteins database, compared to the fugu Takifugu rubripes) proteins database. It was selected sequences of interest genes in the local database of E. marginatus, and all sequences used showed a high similarity degree with another teleosteo species, and were considered adequate to run the real time PCR analyze