Hydrophilic interaction liquid chromatography coupled to negative-ion electrospray linear ion-trap multiple-stage MS (HILIC-ESI-MS , n = 2,3) was used to characterize polar lipids occurring in donkey milk. Besides the detection of abundant phospholipids, the structural characterization and content evaluation of minor glycosphingolipids (GSLs) were assessed. We report an unprecedented characterization of 11 hexosyl-ceramides (HexCer), 10 Hex Cer, and 4 Hex Cer. CID-MS/MS spectra in negative ion mode mainly afford information on the monosaccharide number and ceramide constitution (i.e., N-acyl residue and long-chain base), whereas MS/MS/MS spectra on the ceramide anions allow to recognize for each GSL the sphingoid base. The occurrence of sphingosine (S), sphinganine (DS), and phytosphingosine (P) was inferred from the fragmentation patterns. The milk samples exhibit a relatively high number of phytosphingosine substitutes, perhaps because of the feeding of donkeys, mainly based on pasture grass. However, the incidence of hydroxylated species on the α-carbon of the acyl chain was also revealed. The fatty acid composition of N-acyl chains showed high values of long-chain saturated fatty acids such as 20:0, 22:0, 23:0, and 24:0. An average content of GSL is also provided and three representative mono-, di-, and tri-HexCer in donkey milk are the following: HexCer 18:0/24:1 phytosphingosine nonhydroxylated [PN] at m/z 862.6 as chloride adduct [M+Cl] , and content 225.9 ± 2.8 μg 100 mL ; Hex Cer 18:0/16:0 sphinganine nonhydroxylated [DSN] at m/z 862.7 as deprotonated adduct [M-H] , and content 70.8 ± 1.4 μg 100 mL ; and Hex Cer 18:1/24:1 [SN] at m/z 1132.8 as [M-H] , and content 38.5 ± 0.7 μg 100 mL .