Under nitrogen-depleted conditions, N2-fixing cyanobacteria of the order Nostocales and Stigonematales differentiate vegetative cells into heterocysts. The cell envelope of these specialized cells contains unique glycolipids, consisting of a sugar moiety glycosidically bound to long-chain diols, triols and hydroxyketones. Only few reports have been published on these glycolipids in cultured cyanobacteria and none has reported them in natural environments. Here we show that heterocyst glycolipids can be rapidly and sensitively analyzed using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS2). Positive ion mass spectra of the glycolipids consisted of protonated molecules and diagnostic product ions, indicating losses of sugar groups as well as hydroxyl and carbonyl functionalities from an alkyl chain. Using this method, heterocyst glycolipids were for the first time identified in a natural ecosystem, i.e., a microbial mat from the North Sea barrier island Schiermonnikoog, The Netherlands. This technique will facilitate the quick screening of cyanobacterial cultures and natural environments for the presence of heterocyst glycolipids, which may aid in assessing the role of heterocystous cyanobacteria in the global nitrogen cycle.