<jats:title>Abstract</jats:title><jats:p>Identification of molecular species of various <jats:italic>N</jats:italic>‐acylated bacteriohopanehexol‐mannosamides from the thermophilic bacterium <jats:italic>Alicyclobacillus acidoterrestris</jats:italic> by semipreparative HPLC and by RP‐HPLC with ESI is described. We used triple‐quadrupole type mass spectrometer, <jats:sup>1</jats:sup>H and <jats:sup>13</jats:sup>C NMR for analyzing this complex lipid. CD spectra of two compounds (model compound—7‐deoxy‐<jats:sc>d</jats:sc>‐<jats:italic>glycero</jats:italic>‐<jats:sc>d</jats:sc>‐<jats:italic>allo</jats:italic>‐heptitol obtained by stereospecific synthesis, and an isolated derivative of hopane) were also measured and the absolute configuration of both compounds was determined. On the basis of all the above methods, we identified the full structure of a new class of bacteriohopanes, represented by various <jats:italic>N</jats:italic>‐acylated bacteriohopanehexol‐mannosamides.