BACKGROUND Cardiolipin (CL), a glycerophospholipid containing four acyl chains, is found in most organisms including Gram-negative bacteria such as Escherichia coli. While CL composes only a fraction of the total glycerophospholipids, the four acyl chains lead to a large number of possible molecular species as defined by the total number of carbons and unsaturations in the acyl chains. Understanding the molecular composition of CL, and how it changes under different growth conditions, will aid in understanding the complex role of CL in E. coli. METHODS Normal-phase liquid chromatography/electrospray ionization mass spectrometry was used to quantify the CL molecular species (as defined by the total number of carbons:unsaturations in the acyl chains) in lipid extracts prepared from E. coli grown at 15 °C, 30 °C, 37 °C and 42 °C. RESULTS Fifty-six different CL species were identified as [M-2H](2-) ions in E. coli lipid extracts ranging from 60:0 to 72:4. CL species with an increased total number of unsaturations were more abundant in lipid extracts prepared from cells grown at 15 °C as compared to higher temperatures. CONCLUSIONS This work characterizes the CL composition of E. coli cells grown at various temperatures. By quantifying CL species at a molecular level we have illuminated the molecular complexity of the CL in this relatively simple model organism. This data will be useful for understanding CL function in E. coli and other organisms.