RATIONALE Glycerol serves as the principal backbone moiety bound to various acyl/alkyl chains for membrane lipids of Eukarya , Bacteria , and Archaea . In this study, we report a suite of unusual tetraether lipids in which one of the two conventional glycerol backbones is substituted by butanetriol or pentanetriol. METHODS Identification of these lipids was achieved via diagnostic fragments and their expected acetylation products using liquid chromatography/mass spectrometry (LC/MS), and their diagnostic ether cleavage products using gas chromatography/mass spectrometry (GC/MS). RESULTS We observed structural variations in the polyol backbones and alkyl chains and term these core lipid derivatives: isoprenoidal butanetriol dialkyl glycerol tetraethers (iso‐BDGTs), isoprenoidal pentanetriol dialkyl glycerol tetraethers (iso‐PDGTs), and hybrid isoprenoidal/branched BDGTs and PDGTs (ib‐BDGTs, ib‐PDGTs). Of these, iso‐BDGTs were the most abundant with a methylation at either the sn ‐1 or sn ‐3 position of glycerol and were also found as part of intact polar lipids, adjoined to mono‐ or diglycosidic headgroups. Iso‐BDGTs and iso‐PDGTs are likely produced by Archaea , as indicated by the presence of the characteristic biphytanyl moieties. CONCLUSIONS Butanetriol‐ and pentanetriol‐based tetraether lipids occur in modern estuarine and deeply buried subseafloor sediments, suggesting the presence of alternative backbones in archaeal lipids. Copyright © 2013 John Wiley & Sons, Ltd.