The polar glycolipids were isolated from the thermophilic bacteria Meiothermus taiwanensis ATCC BAA‐400 by ethanol extraction and purified by Sephadex LH‐20 and silica gel column chromatography. The fatty acid composition of O‐acyl groups in the glycolipids was obtained by gas chromatography mass spectroscopy analysis on their methyl esters derived from methanolysis and was made mainly of C15:0 (34.0%) and C17:0 (42.3%) fatty acids, with the majority as branched fatty acids (over 80%). Removal of O‐acyl groups under mild basic conditions provided two glycolipids, which differ only in N‐acyl substitution on a hexosamine. Electrospray mass spectroscopy analysis revealed that one has a C17:0 N‐acyl group and the other hydroxy C17:0 in a ratio of about 1 : 3.5. Furthermore, complete de‐lipidation with strong base followed by selective N‐acetylation resulted in a homogeneous tetraglycosyl glycerol. The linkages and configurations of the carbohydrate moiety were then elucidated by MS and various NMR analyses. Thus, the major glycolipid from M. taiwanensis ATCC BAA‐400 was determined with the following structure: α‐Galp(1‐6)‐β‐Galp(1‐6)‐β‐GalNAcyl(1,2)‐α‐Glc(1,1)‐Gro diester, where N‐acyl is C17:0 or hydroxy C17:0 fatty acid and the glycerol esters were mainly iso‐ and anteisobranched C15:0 and C17:0.