Hexactinellid sponges are often considered to be the most ancient metazoans. Lipid biomarkers from 23 species were studied for information on their phylogenetic properties, particularly their disputed relation to the two other sponge classes (Demospongiae, Calcarea). The most prominent lipid compounds in the Hexactinellida comprise C28 to C32 polyenoic fatty acids. Their structures parallel the unique patterns found in demosponge membrane fatty acids (demospongic acids) and strongly support a close phylogenetic association of the Demospongiae and the Hexactinellida. Both taxa also show unusual mid-chain methylated fatty acids (C15 C25 ) and irregular C25- and C40-isoprenoid hydrocarbons, tracers for specific eubacteria and Archaea, respectively. These biomarkers indicate a similar, highly conservative symbiont community, although some shift in the abundance of the associated microbiota was observed. The lack of these features in calcareous sponges further contradicts the still common view that Calcarea and Demospongiae are more closely related to each other than either is to the Hexactinellida.