Variation in brain FA composition, particularly decreased DHA (22:6n-3), affects neurodevelopment, altering visual, attentional, and cognitive functions, and is implicated in several neuropsychiatric disorders. To further understand how specific brain processes and systems are affected by variation in brain DHA content, we sought to determine whether specific brain regions were differentially affected by treatments that alter brain DHA content. Adult male Long-Evans rats were raised from conception using diet/breeding treatments to produce four groups with distinct brain phospholipid compositions. Total phospholipid FA composition was determined in whole brain and 15 brain regions by TLC/GC. Brain regions exhibited significantly different DHA contents, with the highest levels observed in the frontal cortex and the lowest in the substantia nigra/ventral tegmental area. Increased availability of DHA resulted in increased DHA content only in the olfactory bulb, parietal cortex, and substantia nigra/ventral tegmental area. In contrast, treatment that decreased whole-brain DHA levels decreased DHA content in all brain regions except the thalamus, dorsal midbrain, and the substantia nigra/ventral tegmental area. Alterations in DHA level were accompanied by changes in docosapentaenoic acid (n-6 DPA, 22:5n-6) content; however, the change in DHA and n-6 DPA was nonreciprocal in some brain regions. These findings demonstrate that the FA compositions of specific brain regions are differentially affected by variation in DHA availability during development. These differential effects may contribute to the specific neurochemical and behavioral effects observed in animals with variation in brain DHA content.