Phosphatidylcholine (PC), a major lipid class in the membranes of eukaryotes, is synthesized either via the triple methylation of phosphatidylethanolamine (PE) or via the CDP-choline route. To investigate whether the two biosynthetic routes contribute differently to the steady-state profile of PC species, i.e., PC molecules with specific acyl chain compositions, the pools of newly synthesized PC species were monitored by labeling Saccharomyces cerevisiae with deuterated precursors of the two routes, (methyl-D3)-methionine and (D13)-choline, respectively. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) revealed that the two PC biosynthetic pathways yield different sets of PC species, with the CDP-choline route contributing most to the molecular diversity. Moreover, yeast was shown to be capable of remodeling PC by acyl chain exchange at the sn-1 position of the glycerol backbone. Remodeling was found to be required to generate the steady-state species distribution of PC. This is the first study demonstrating a functional difference between the two biosynthetic routes in yeast.