From the point of view of the origin of life, it is necessary to clarify the importance of membrane lipids in early evolutionary times. It is assumed that the original microorganisms lived in environments similar to hydrothermal vents and crust fissures or volcanoes. The ancient (supposedly 35,000 years old when reaching the surface) underground waters of the famous spa town of Karlovy Vary (Carlsbad), Czech Republic, represent an ideal source of thermotolerant bacteria potentially relevant to the study of early evolutionary times. We studied bacterial isolates from these thermal springs and their physiological adaptation to the lowest and highest cultivation temperatures. Two different temperatures illustrate the climatic changes that occurred between the origin of life and the time when the Earth began to cool. In this study, we have reported for the first time the presence of four PG isomers (i.e. R,S, R,R, S,R, and S,S) identified by chiral chromatography in five thermotolerant bacteria (Table in Supplementary Information) at different concentrations. The assumption stated in many papers that archaea versus bacteria and eukaryotes produce only one isomer of PG was not confirmed.