A non-aqueous reversed phase high performance liquid chromatography method was developed, and optimized for triacylglycerol analysis in a Brevoortia tyrannus (menhaden) oil sample. Four columns were serially coupled to tackle such a task, for a total length of 60cm of shell-packed stationary phase, and operated under ultra high pressure conditions. As detection, positive-ion atmospheric pressure chemical ionization mass spectrometry was used to attain identification of the analyzed sample components. A number of 137 triacylglycerols containing up to 19 fatty acids, with 14–22 carbon atom alkyl chain length and 0–6 double bonds, were positively identified in the complex lipidic sample. This is the first work that reports an extensive characterization of the triacylglycerol fraction of menhaden oil.