Docosahexaenoic acid (DHA) is mostly esterified in food and is easily oxidized by exposure to heat or light. Hydroperoxide positions of DHA mono-hydroperoxide (DHA;OOH) provide information on oxidation mechanisms (e.g., radical- or singlet oxygen oxidation), yet direct identification of esterified DHA;OOH isomers has not been achieved. We previously accomplished the direct analysis of free DHA;OOH isomers with liquid chromatography-mass spectrometry (LC-MS/MS). In this study, we developed an LC-MS/MS method for direct analysis of esterified DHA;OOH based on our previous study. The developed method was capable of distinguishing esterified DHA;OOH isomers in raw- and oxidized mackerel. The result suggested that radical oxidation of esterified DHA can progress even in refrigeration. Different transitions were observed depending on the oxidation mechanism and lipid class. The analytical method and insights obtained in this study would be valuable to further understand and effectively prevent DHA oxidation in food products.