Aerobic methane-oxidizing bacteria (MB) are the primary terrestrial sinks for the greenhouse gas methane. A distinct characteristic of MB is the presence of specific phospholipid ester-linked fatty acids (PLFA) in their membranes that differentiate them from each other and also from all other organisms. These distinct PLFA patterns facilitate microbial ecology studies. For example, the assimilation of C from methane into PLFA can be traced in environmental samples using stable isotope (13C) probing (SIP), which links the activity of MB to their community composition in situ. However, the phylogenetic resolution of this method is low because of a lack of PLFA profiles from cultured MB species. In this study, PLFA profiles of 22 alphaproteobacterial (type II) MB were analysed after growth on methane, methanol or both substrates together. Growth on different substrates did not affect the PLFA profiles of the investigated strains. A number of Methylocystis strains contained novel C18:2 fatty acids (7c,12c and 6c,12c) that can be used as diagnostic biomarkers. The detection of these novel PLFA, combined with the analyses of multiple type II strains, increased the phylogenetic resolution of PLFA analysis substantially. Multivariate analysis of the expanded MB PLFA database identified species groups that closely reflected phylogenies based on 16S rRNA and pmoA gene sequences. The PLFA database therefore provides a robust framework for linking identity to activity in MB communities with a higher resolution than was previously possible.