Studies of the distribution of macroamphiphiles in Gram-positive bacteria are interesting in relation to understanding their functions and are of chemotaxonomic value at the supergeneric level. Previous studies have revealed the distribution of lipoteichoic acid (LTA) in the low G+C phylum Firmicutes and typically parallel that of the teichoic acid as a secondary cell wall polymer (SCWP). The present study had focused on the distribution of macroamphiphiles in the high G+C phylum Actinobacteria, where most previous studies have revealed liopglycans as the macroamphiphiles in various different lineages of the phylum. The present study has, for the first time, investigated macromaphiphiles in a thermophilic Actinobacterium, Thermobifida fusca. The study detected the presence of LTA, with detailed structural analysis. This confirms the compatibility of these macromolecules with membrane adaptation at higher temperatures. A second thermophilic Actinobacteria, Rubrobacter xylanophilus (which had moved recently to the most distant lineage of the phylum) was found to lack of typical macroamphiphiles. The present study also detected a novel LAM-like molecule in Kineococcus radiotolerans suggesting a close relationship between the distribution of LAM or LAM-like molecules and that of the SCWP, arabinogalactan (AG). The study also identified the presence both LTA and lipoglycan in two representatives of the genus Streptomyces (Streptomyces coelicolor and Streptomyces sp. DSM40537). This allows a re-evaluation of the hypothesis (Fischer, 1994) that a single type of major macroamphiphile is normally present in a single organism. Extending these findings, comparative genomic analyses suggest that the LTA biosynthesis pathway in Actinobacteria might be different from that in the Firmicutes. The alanine substitution pathway for LTA and teichoic acids (TA) was also found to be absent from the phylum Actinobacteria. Moreover, the comparative genomic analyses for Kineoccus radiotolerans were consistent with the practical results for this organism, illustrating the potential of predicting macroamphiphile composition utilizing genome databases. The study had also confirmed that the distribution of macroamphiphiles and SCWPs has importance chemotaxonomic value especially at the supra-generic level; it can be hypothesised from the study that: LTA containing Actinobacteria contain TA as a SCWP, whilst lipoglycan containing Actinobacteria generally contains other SCWPs, such as AG rather than TA.