There are several possible pathways for the biosynthesis of PG isomers in biological systems, but it is not yet clear which of them are currently in use. In this study, we analyzed seven strains of thermotolerant bacteria by chiral HPLC and, for the first time, detected all four PG isomers in the five of the bacteria examined. Although there are many studies on the molecular species composition and physiological functions of PGs from microorganisms, only a few studies distinguish between stereoisomers. Details on the distribution of PG isomers in microorganisms and differences in metabolic and physiological functions between stereoisomers can be expected to be elucidated in the future. Based on the results of the analysis presented in this paper and the articles mentioned above, it can be assumed that with the development of instrumental analytical techniques and the progress of whole genomes, especially for bacteria isolated from extreme environments, it will gradually be clarified.