High performance liquid chromatography-electrospray ionization tandem mass spectrometry was applied to the comprehensive analysis of phospholipids from seven Antarctic and seven non-Antarctic yeasts. Identification of specific fatty acyl moieties to the sn-1 and sn-2 positions of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol (PI) were determined by relative abundance of fragment ions associated with formation of carboxylate anions and loss of fragment ions as free fatty carboxylic acid and ketene. Modulations with growth temperature in fatty acyl moieties in the sn-1 and sn-2 positions were characterized. Principal component analysis demonstrated that PE, PC and to a lesser extent PS, but not PI, were grouped into three distinct clusters consisting of seven Antarctic yeasts (Cryptococcus victoriae, Holtermanniella wattica, H. nyarrowii, Candida psychrophila, Leucosporidium fellii, Glaciozyma antarctica, Rhodotorula mucilaginosa), four non-Antarctic yeasts (C. albicans, Zygosaccharomyces rouxii, Cr. humicolus, R. mucilaginosa) and three strains of Saccharomyces cerevisiae. •Development of electrospray ionization-mass spectrometry for yeast phospholipids.•Assignment of specific fatty acyl moieties to sn-1 and sn-2 positions.•The method is highly sensitive and reproducible.•Antarctic and non-Antarctic yeasts clearly differentiated.