Recently, we demonstrated that tandem mass spectrometry (MS/MS) analysis in the presence of sodium ions was useful for identification of the position of the hydroperoxy group in phosphatidylcholine hydroperoxide (PCOOH). Likewise, MS/MS may enable identification of the hydroperoxy group position in various lipid hydroperoxides (LOOHs). To this end, we prepared major LOOHs, namely hydroperoxyoctadecadienoic acid (HPODE) and hydroperoxyeicosatetraenoic acid (HPETE), and analyzed them by quadrupole-time-of-flight MS/MS in both the absence and presence of alkali metals. Photo-oxidation (singlet oxygen-induced oxidation) of linoleic acid (LA) was used to prepare 9-10E,12Z-HPODE, 9-10E,12E-HPODE, 10-8E,12Z-HPODE, 12-9Z,13E-HPODE, 13-9Z,11E-HPODE, and 13-9E,11E-HPODE. Each isomer was analyzed under various MS/MS conditions (e.g., absence and presence of sodium). We found that in the presence of alkali metals, especially sodium, collision-induced dissociation (CID) of all HPODE isomers yielded structure-diagnostic fragment ions that were highly useful in identifying the position of the hydroperoxy group. For instance, CID spectra of sodiated 13-9Z,11E-HPODE revealed a neutral loss of 88 Da arising from fragmentation of the hydroperoxy group. Similar results were observed for HPETE isomers. Following oxidation of LA (or arachidonic acid) by lipoxygenase, the hydroperoxy group position of the resultant HPODE (or HPETE) was easily identified using this method, without any chromatographic separation processes. As information on the position of the hydroperoxy group provides insight into the processes that initiate lipid peroxidation (e.g., enzymatic oxidation, auto-oxidation and singlet oxygen-induced oxidation), the proposed method may be useful in elucidating the involvement and mechanism of lipid peroxidation in food deterioration and pathophysiological processes.