A Siberian peat contains a high abundance of three unknown isomers of hexamethylated branched (br) GDGTs, of which two are present as a second peak eluting shortly after the known hexamethyl br GDGT containing two 5,13,16-trimethyloctacosanyl moieties. Tandem high performance liquid chromatography–mass spectrometry (HPLC–MS2) did not reveal substantial differences in the MS2 spectrum of the two peaks. HPLC isolation of the two peaks of the hexamethylated br GDGT followed by ether cleavage and GC–MS characterization of the hydrocarbons formed, using supersonic molecular beam (SMB) ionization, revealed the second peak to be composed mainly of a novel hexamethyl br GDGT, possessing two 6,13,16-trimethyloctacosanyl moieties. In addition, both chromatographically separated hexamethylated br GDGTs contained a smaller quantity (20–33%) of a non-symmetrical isomer, with one 13,16-dimethyloctacosanyl and one tetramethyloctacosanyl (either 5,13,16,24- or 6,13,16,23-) moiety. Hexamethylated br GDGTs thus consist of four structural isomers. Furthermore, the pentamethylated br GDGT was also isolated and shown to contain both 5,13,16- and 6,13,16-trimethyloctacosanyl moieties, so is composed of two structural isomers. The assignments reveal why environmental distributions of br GDGTs are sometimes complex, which may have implications for the use of br GDGTs in palaeoclimate reconstruction.