Spatial changes of fatty acids in the retina in response to different dietary n-3 formulations have never been explored, although a diet rich in EPA and DHA is recommended to protect the retina against the effects of aging. In this study, Wistar rats were fed for 8 weeks with balanced diet including either EPA-containing phospholipids, EPA-containing triglycerides, DHA-containing phospholipids, or DHA-containing triglycerides. Qualitative changes in fatty acid composition of plasma, erythrocytes and retina were evaluated by gas chromatography-flame ionization detector. Following the different dietary intakes, changes to the quantity and spatial organization of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species in retina were determined by liquid chromatography coupled to tandem mass spectrometry and MALDI (Matrix Assisted Laser Desorption Ionization) coupled to mass spectrometry imaging. The omega-3 content in the lipids of plasma and erythrocytes suggests that phospholipids as well as triglycerides are good omega-3 carriers for retina. However, a significant increase in DHA content in retina was observed, especially molecular species as di-DHA-containing PC and PE, as well as an increase in very long chain polyunsaturated fatty acids (VLC-PUFAs, more than 28 carbons) following phospholipid-EPA and triglyceride-DHA diets only. All supplemented diets triggered spatial organization changes of DHA in the photoreceptor layer around the optic nerve. Taken together, these findings suggest that dietary omega-3 supplementation can modify the content of fatty acids in the rat retina.