Mayflies (Ephemeroptera) in aquatic naiad stages are known to be filter feeders of detritus and algae, but are nonfeeding during their short adult life. Members of Hexagenia form U-shaped burrows in the silt of lake bottoms or streams where they remain for a year or more before emerging as subimagos to fly to the shore. For activities such as mating flights and oviposition, adults must rely on stored energy sources which are principally glycogen and lipids. This study focuses on the fatty acid composition of lipid stores in Hexagenia limbata (Serville) adults and late-instar naiads from a North Central Texas lake. Lipids from whole-body homogenates were extracted with organic solvents, and fatty acids were analyzed by gas chromatography-mass spectrometry (GC-MS). Total lipid extracts, as well as neutral lipids (triglycerides and diglycerides) and polar lipids (phospholipids), were analyzed. Adult males exhibited the highest amount of lipid as a percentage of dry weight, followed by naiads and females. The most abundant fatty acids were 16∶0 > 18∶1 > 16∶1 in both naiads and males and 18∶1 > 16∶0 > 16∶1 in females. Phospholipids and triglycerides were the most prominent lipid classes in naiads while free fatty acids were abundant in males, which also showed a diglyceride fraction. Triglycerides were the main lipid fraction in females. The profiles suggest that dietary fatty acids are stored in naiad triglycerides, and once adult emergence occurs, the triglycerides appear to be retained in the females primarily for oocyte production; whereas they are quickly mobilized to supply energy for flight in the males.