Little is known about the lipid composition of cold-adapted dinoflagellates. Here, we investigate Peridinium aciculiferum and Scrippsiella hangoei, two cold-adapted dinoflagellate morphospecies that present significant differences in general morphology, physiology and habitat, but which share identical ribosomal DNA (rDNA) sequences, indicating a recent evolutionary divergence between them. Despite their present taxonomic names, it is now known that these two genetically isolated morphospecies are evolutionarily related to the so-called Pfiesteria-like species. We have found that chloroplastassociated glycolipids from P. aciculiferum and S. hangoei contained a higher relative percentage of two polyunsaturated C 18 fatty acids (octadecapentaenoic [18:5(n-3)] and octadecatetraenoic [18:4(n-3)] acids), and a lower relative percentage of hexadecanoic acid (16:0) than typically observed in previously examined warm-adapted dinoflagellate species. This suggests that these two fatty acids provided the requisite membrane fluidity for chloroplasts under a cold growth condition in these two organisms. A similar increase in 18:5(n-3) and 18:4(n-3) over warm-adapted species was also found in the phospholipids and triglycerides. P. aciculiferum and S. hangoei were also examined for the production of unique sterols that may serve as chemical biomarkers. Both were found to have a very similar sterol composition, consisting of two common dinoflagellate sterols, cholestanol and dinostanol, as dominant sterols, along with a number of other common dinoflagellate sterols as minor sterols. This demonstrated the absence of a specific steroidal biomarker but, more importantly, showed that these two morphospecies still produce very similar sterols despite having diverged in several other traits.