Edible fats and oils are mainly composed of triacylglycerols synthesized by plants and animals. Natural triacylglycerols are predominantly such complex mixtures that it is impossible to separate all molecules by a single analytical technique. However, the molecular structure of triacylglycerols is of great importance from the biochemical, nutritional, and technological points of view. This article reviews the chromatographic and mass spectrometric methods in the analysis of triacylglycerols as well as the techniques for stereospecific analysis, with special attention to the analysis of milk fat and fish oils. The principles of triacylglycerol separation with high‐performance liquid chromatographic, gas chromatographic, and supercritical fluid chromatographic methods are described. In general, chromatographic techniques offer a wide variety of possibilities for separation of molecular species of triacylglycerols, but the identification of the components is often a problem. Mass spectrometry provides information both for structure elucidation and for quantitation purposes. The advantages of different ionization modes in the analysis of triacylglycerols are presented. In addition, the usefulness of tandem mass spectrometric methods, yielding information on the molecular association of fatty acids and the regiospecific distribution of fatty acids(sn‐2 andsn‐1/3 positions) is discussed. The traditional methods for the complete stereospecific analysis of triacyl‐sn‐glycerols include the phosphorylation of diacylglycerols and their hydrolysis with phospho‐lipases. Over the last few years methods for the separation of enantio‐meric acylglycerols have been developed, and their capability for the stereospecific analysis of triacylglycerols is discussed in this review.