Unusual shorter-chain alkenones (C 35 and C 36 ) were the dominant alkenones (rather than C 37 and C 38 ) isolated from commercially grown Tisochrysis lutea microalgae. Structures of the alkenones were determined by analyzing the alkenones and their products following a butenolysis reaction by comprehensive two-dimensional gas chromatography coupled to a high-resolution time-of-flight mass spectrometer (GC × GC-TOF HRMS). The major alkenones were identified as (15E,22E)-heptatriaconta-15,22-dien-2-one and (16E,23E)-octatriaconta-16,23-dien-3-one, similar to one previous finding of abundant shorter-chain alkenones in a culture of Emiliania huxleyi CCMP2758, yet distinguishing them from C 35 and C 36 alkenones extracted from Black Sea sediments with different double bond positions. Our data suggest a possible shared trigger (e.g. nutrient stress) that may occur during cultivation resulting in shorter-chain alkenones from both the aforementioned E. huxleyi and T. lutea. Moreover, for T. lutea, short-chain alkenone biosynthesis can be accompanied by lower alkenone content but higher amounts of valuable docosahexaenoic acid (DHA). These results, therefore, extend beyond alkenones and are important to established and emerging algal oil-based industries.Keywords Isochrysis Á Tisochrysis Á Alkenones Á Omega-3 fatty acids Á Butenolysis Á Comprehensive twodimensional gas chromatography