Evidence from 31P magnetic resonance spectroscopy (31P MRS) studies suggest that different psychiatric disorders, which typically emerge during adolescence and young adulthood, are associated with abnormalities in mitochondrial bioenergetics and membrane phospholipid metabolism. These disorders are also associated with deficits in omega-3 polyunsaturated fatty acids (n-3 PUFA), including docosahexaenoic acid (DHA) which accumulates in mitochondrial and synaptic membranes. The present study investigated the effects of dietary-induced alterations in brain DHA accrual during adolescence on phospholipid metabolism and bioenergetics in the adult rat brain using 31P MRS. During the peri-adolescent period (P21-P90), male rats were fed a diet with no n-3 fatty acids (Deficient, DEF, n = 20), a diet fortified with preformed DHA (fish oil, FO, n = 20), or a control diet fortified with alpha-linolenic acid (18:3n-3, n = 20). On P90, 31P MRS was performed under isoflurane anesthetic using a 7 T Bruker Biospec system. Compared with controls, brain DHA levels were significantly lower in adult rats fed the DEF diet (-17%, p ≤ 0.0001) and significantly higher in rats fed the FO diet (+14%, p ≤ 0.0001). There were no significant group differences for indices of bioenergetics, including adenosine triphosphate and phosphocreatine levels, or indices of membrane phospholipid metabolism including phosphomonoesters and phosphodiesters. Therefore, the present 31P MRS data suggest that rat brain DHA levels are not a significant predictor of mitochondrial bioenergetics or membrane phospholipid metabolism.