Yeast is a poikilothermic organism and adapts its lipid composition to the environmental temperature to maintain membrane physical properties. Studies addressing temperature-dependent adaptation of the lipidome have described changes in the phospholipid composition at the level of sum composition (e.g. PC 32:1) and molecular composition (e.g. PC 16:0_16:1). However, there is little information at the level of positional isomers (e.g. PC 16:0/16:1 versus PC 16:1/16:0). Here, we used collision- and ozone-induced dissociation (CID/OzID) mass spectrometry to investigate homeoviscous adaptation of PC, PE and PS to determine the phospholipid acyl chains at the sn-1 and sn-2 position. Our data establish the sn-molecular species composition of PC, PE and PS in the lipidome of yeast cultured at different temperatures.