Structural analogs of the bioactive lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol were synthesized with a xylitol polar head group and both diacyl and diether radyl groups. Mass spectral characterization of xylitol phospholipids (PX) was carried out using collisional activation and high-resolution mass measurements of positive molecular ion species and compared with the phosphatidylglycerol (PG) analogs. PX were synthesized using a transphosphatidylation reaction catalyzed by phospholipase D and purified by high-performance liquid chromatography (HPLC). Compounds were subjected to electrospray ionization and collision-induced dissociation (CID) was performed using a tandem quadrupole mass spectrometer to generate positive and negative molecular ions. Diether phospholipids were additionally analyzed by high-resolution mass spectrometry as protonated and sodiated molecular species in positive ion mode. Ester-linked PX analogs behaved similarly to PG after collisional activation of [M - H]- . The product ions formed by CID of the diether PG and PX negative ions only revealed information about the head group with no information about the aliphatic chains. In contrast, CID of protonated and sodiated diether phospholipid positive ions revealed reactions corresponding to cleavage of the ether chain, likely occurring by charge-driven reaction mechanisms. Novel PX analogs with diacyl and diether radyl substituents of the glycerol backbone were characterized by tandem mass spectrometry. These unique diether phospholipid analogs enabled exploration of ether cleavage reactions of the positive molecular ion species resulting from collision-induced decomposition. Copyright © 2016 John Wiley & Sons, Ltd.