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The composition of the core lipids and intact polar lipids (IPLs) of five Rubrobacter species was examined. Methylated (ω-4) fatty acids (FAs) characterized the core lipids of Rubrobacter radiotolerans, R. xylanophilus and R. bracarensis. In contrast, R. calidifluminis and R. naiadicus lacked ω-4 methyl FAs but instead contained abundant (i.e., 34-41 % of the core lipids) ω-cyclohexyl FAs not reported before in the order Rubrobacterales. Their genomes contained an almost complete operon encoding proteins enabling production of cyclohexane carboxylic acid CoA thioester, which acts as a building block for ω-cyclohexyl FAs in other bacteria. Hence, the most plausible explanation for the biosynthesis of these cyclic FAs in R. calidifluminis and R. naiadicus is a recent acquisition of this operon. All strains contained 1-O-alkyl glycerol ether lipids in abundance (up to 46 % of the core lipids), in line with the dominance (>90 %) of mixed ether/ester IPLs with a variety of polar headgroups. The IPL head group distribution of R. calidifluminis and R. naiadicus differed, e.g. they lacked a novel IPL tentatively assigned as phosphothreoninol. The genomes of all five Rubrobacter species contained a putative operon encoding the synthesis of the 1-O-alkyl glycerol phosphate, the presumed building block of mixed ether/ester IPLs, which shows some resemblance with an operon enabling ether lipid production in various other aerobic bacteria but requires more study. The uncommon dominance of mixed ether/ester IPLs in Rubrobacter species exemplifies our recent growing awareness that the lipid divide between archaea and bacteria/eukaryotes is not as clear cut as previously thought., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)