Thirty-four axenic strains of cyanobacteria were analysed for their glycolipid content using high performance liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (HPLC/ESI-MS2). Species of the families Nostocaceae and Rivulariaceae, capable of biosynthesising heterocysts, contained a suite of glycolipids consisting of sugar moieties glycosidically bound to long-chain diols, triols, keto-ols and keto-diols. The aglycone moiety consisted of C26 or C28 carbon-chains with hydroxyl groups at the C-3, ω-1 or ω-3 positions. Keto-ols and keto-diols contained their carbonyl functionalities likely at the C-3 position. These compounds were absent in all analysed unicellular and filamentous non-heterocystous cyanobacteria and in the heterocyst-forming cyanobacterium Anabaena CCY9922 grown in the presence of combined nitrogen, supporting the idea that the long-chain glycolipids are an important and unique structural component of the heterocyst cell envelope. The glycolipids 1-(O-hexose)-3,25-hexacosanediol and 1-(O-hexose)-3-keto-25-hexacosanol were ubiquitously distributed in species of the family Nostocaceae. 1-(O-hexose)-3,25,27-octacosanetriol and 1-(O-hexose)-3-keto-25,27-octacosanediol were dominant in members of the Calothrix genus, while traces of those compounds were detected only in one species of the Nostocaceae family. Their distribution in heterocystous cyanobacteria suggests a chemotaxonomic relevance that might allow distinguishing between species of different genera. Culture experiments indicate that the amount of keto-ols and keto-diols decreases relatively to their corresponding diols and triols counterparts with increasing temperature. Possibly, this is an adaptation to optimise the cell wall gas permeability, preventing inactivation of the oxygen-sensitive nitrogenase while allowing the highest diffusion of atmospheric dinitrogen into the heterocyst.