High resolution electrospray ionization tandem mass spectrometry (HR-ESI-MS/MS) was used to analyze cardiolipins (Ptd2Gro) including their plasmalogen forms from three species of the anaerobic beer-spoilage bacterial genus Pectinatus. Cardiolipins including their plasmalogens were analyzed by HR-ESI-MS/MS on Orbitrap and almost 100 cardiolipins (i.e. tetra-acyl--Ptd2Gro, plasmenyl-tri-acyl--PlsPtd2Gro, and di-plasmenyl-di-acyl--Pls2Ptd2Gro) of three classes were identified. The structures of the molecular species that consist of various regioisomers and structurally similar compounds were revealed in detail. The high resolution mass spectrometry allowed the unambiguous structural assignment of Ptd2Gro, PlsPtd2Gro, and Pls2Ptd2Gro in the three species of Pectinatus, which contain predominantly odd numbered fatty acids.