Our objective was to identify and quantify phospholipids in milk from different species (human HM, cow CoM, camel CaM, mare MM) using an optimised 31P NMR spectroscopy procedure. The phospholipid fingerprints were species-specific with a broader variety of classes found in HM and MM; HM and CaM were richer in sphingomyelin (78.3 and 117.5 µg/ml) and plasmalogens (27.3 and 24 µg/ml), possibly important for infant development. Total phospholipid content was higher in CaM (0.503 mM) and lower in MM (0.101 mM) compared to HM (0.324 mM) or CoM (0.265 mM). Our optimised method showed good sensitivity, high resolution, and easy sample preparation with minimal loss of target molecules. It is suitable for determining the accurate composition of a large number of bioactive phospholipids with putative health benefits, including plasmalogens, and should aid in selecting appropriate ingredient sources for infant milk substitutes or fortifiers, and for functional foods dedicated to adults.