The lipid composition of Roseiflexus castenholzii, a thermophilic filamentous phototrophic bacterium related to uncultivated filamentous phototrophic bacteria that predominate in hot spring microbial mats, is reported. R. castenholzii lipid extracts were dominated by components characterized by alkane-1-ol-2-alkanoate moieties glycosidically bonded to a C6 sugar. Similar fatty glycosides, with an additional fatty acid esterified, were detected by HPLC-MS. R. castenholzii also produces a suite of wax esters ranging from 37 to 40 carbon atoms in length. In lipid extracts from two nonsulfidic hot spring microbial mats, similar alkane-1,2-diol-based lipids were detected in minor amounts. R. castenholzii lipids are compared to lipids of mats and other thermophilic mat isolates.