Analysis of fatty acids in the form of fatty acid methyl esters (FAMEs) using gas chromatography (GC) is routine within microbiology but still some compounds remain unidentified. During characterization of the FAMEs of two strains of the microaerophilic bacterium Lutibacter sp., recently isolated from the Black Sea, a series of compounds, eluting after the regular FAMEs, were detected. We identified these compounds using GC–mass spectrometry (GC–MS) and an authentic standard, to be amino acids glycine-linked via an amide bond to β-hydroxy fatty acids (i.e. glycineβ-hydroxy fatty acid amides). Analysis of the intact polar lipids of the Lutibacter species by ultra-high performance liquid chromatography–high resolution mass spectrometry (UHPLC–HRMS) showed that the glycine β-hydroxy fatty acid amides are derived from glycine lipids (also known as cytolipins), which are amino acid lipids. Amino acid lipids represent an under-studied, but potentially significant, group of microbial membrane lipids and our results provide a rapid way to detect the presence of glycine lipids during routine fatty acid analysis by GC. Furthermore, glycine β-hydroxy fatty acid amides represent easily detectable biomarker lipids for glycine lipid-producing microorganisms in natural environments.