In this work, ultra high performance liquid chromatography was used for the characterization of non polar lipids (triacylglycerols) in milk samples of different origin, as well as milk-derivatives. For tackling such a task, three core–shell type octadecylsilica columns were serially coupled, reaching a total stationary phase length of 45cm, using acetonitrile-isopropanol gradient elution allowing triacylglicerol separation according to increasing partition number. The employment of an ion-trap-time-of-flight detection in conjunction with atmospheric-pressure chemical ionization mass spectrometry was carried out to positively identify a number of 243 different triacylglycerols containing up to 22 fatty acids, with 2–22 carbon atom alkyl chain length, and 0–3 double bonds. This work reports an extensive characterization of the triacylglycerol fraction in milk and milk-related samples of different sources.