In this work, a method for the separation of triacylglycerols (TAGs) present in human milk and other mammalian species by reversed-phase high performance liquid chromatography using a core-shell particle packed column with UV and evaporative lightscattering detectors is described. Under optimal conditions, a mobile phase containing acetonitrile/n-pentanol at 10 ºC gave an excellent resolution between more than 50 TAG peaks. A small-scale method for fat extraction in these milks (particularly of interest for human milk samples) using minimal amounts of sample and reagents was also developed.The proposed extraction protocol and the traditional method were compared, giving similar results, with respect to the total fat and relative TAG contents. Finally, a statistical study based on linear discriminant analysis on the TAG composition of different types of milks (human, cow, sheep and goat) was carried out to differentiate the samples according to its mammalian origin.