Liquid chromatography coupled to atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) mass spectrometry (MS), in parallel, was used for simultaneous detection of bovine milk sphingolipids (BMS). APCI-MS mass spectra exhibited mostly ceramide-like fragment ions, [Cer-H2O + H]+ and [Cer-2H2O + H]+, which were used to identify individual molecular species of BMS according to fatty acyl chain length:degree of unsaturation and long-chain base (LCB). ESI-MS was used to confirm the molecular weights of BMS species. Both sphingomyelin (SM) and dihydrosphingomyelin (DSM) molecular species were identified, with DSM species constituting 20% of BMS. Approximately 56 to 58% of DSM species contained a d16:0 LCB, while 34 to 37% contained a d18:0 LCB. Approximately 26 to 30% of SM species contained a d16:1 LCB, while 57 to 60% contained a d18:1 LCB. BMS species contained both odd and even carbon chain lengths. The most abundant DSM species contained a d16:0 LCB with a 22:0, 23:0 or 24:0 fatty acyl chain, while the most abundant SM species contained a d18:1 LCB with a 16:0 or 23:0 fatty acyl chain. 31P NMR spectroscopy was used to conclusively confirm that DSM is a dietary component in BMS.