A lipidomic evaluation was performed on the tilapia muscle, head and viscera, including studying the composition, distribution and stereospecific characteristics of fatty acids and lipid species. The head and viscera lipids were significantly richer than the muscle lipids. Triacylglycerols were the predominant fraction (over 80% of total lipid in the muscle and head). Additionally, polyunsaturated fatty acids had higher percentages in phospholipids (30.35–52.05% of total fatty acids) than in triacylglycerols (18.11–25.15%). The C52:2 and C52:3 were the most abundant triacylglycerols, which indicates the potential application in infant food. Moreover, 622, 530 and 513 lipids were identified using ultraperformance liquid chromatography-quadrupole time-of-flight-mass spectrometry in the muscle, head and viscera, respectively. The three tilapia parts were distinguished using multivariate analysis. Five fatty acids and 33 lipid species were considered as the potential biomarkers. This comprehensive analysis will help to evaluate the lipid nutritional values and facilitate exploitation in tilapia consumption and processing.