A new archaeal lipid biomarker, C _20,25 sn-2, C-7 hydroxy isoprenoid glycerol diether, has been identified in methane-related carbonates from six methane seep locations. The molecular structure resembles that of sn-2 hydroxyarchaeol but the hydroxy group-bearing moiety at the sn-2 position comprises a regular C ₂₅ instead of a C ₂₀ isoprenoid chain. Compound specific stable carbon isotope measurements revealed δ ¹³C values ranging from -96‰ to -129‰, which are similar to δ ¹³C values of sn-2 hydroxyarchaeol. This supports the origin of the novel compound from archaea involved in the anaerobic oxidation of methane. It also suggests a common source, i.e. archaea within the ANME-2 group, which is consistent with the previously reported 16S rDNA data for the same samples. The frequent appearance of the novel sn-2 isoprenoidal hydroxy glycerol diether in cold seepage sediments with high salinity environments, and its distinct structural similarities with membrane lipids of halophilic and methanogenic microbes, suggests that ANME-2 methanotrophic archaea thriving at elevated salinity are the possible biological source for this specific lipid.