RATIONALE Most of the liquid chromatography/mass spectrometry (LC/MS) methods that have been developed for the analysis of Diarrhetic Shellfish Poisoning (DSP) toxins in shellfish and algae samples have been unable to differentiate the isomers okadaic acid (OA) and dinophysistoxin‐2 (DTX2), unless separated by chromatography. Since there are many bioconversion products of these compounds it is imperative to determine characteristic product ions, which can provide unequivocal identification of OA and DTX2 and their analogs. METHODS Using electrospray ionization, the fragmentation processes for two types of precursor ions, [M+Na] + and [M–H] – , of the polyether marine toxins, dinophysistoxins (DTXs), were studied using a hybrid linear ion trap Orbitrap mass spectrometer which provided high mass accuracy data in combination with multiple tandem mass (MS n ) spectra. Three structurally related toxins were compared; okadaic acid (OA), dinophysistoxin‐2 (DTX2) and dinophysistoxin‐1 (DTX1). A quick multiple reaction monitoring (MRM) LC/MS/MS method was developed utilizing the characteristic precursor/product ion mass transitions. RESULTS Comparison of the high‐resolution product ion, [M–H] – , spectra of these toxins featured dominant signals that resulted from two six‐centered rearrangements and previously proposed fragmentation pathways for the ion of m/z 321 and 293 have been corrected and identified. By contrast, the [M+Na] + product ion spectra only revealed distinctive ions for the isomers, OA ( m/z 595, 443 and 151) and DTX2 ( m/z 581, 429 and 165). To illustrate the benefits of this study, a mass selective LC/MS/MS method was developed in which the isomers OA and DTX2 co‐eluted but were distinguished using the mass transitions, m/z 827/595, 827/443 (OA) and m/z 827/581, 827/429 (DTX2). CONCLUSIONS Comparison of OA, DTX2 and DTX1 led to the correction of proposed negative ion mode fragmentation pathways. Through extensive study and comparison of the [M+Na] + product ion spectra, distinctive product ions were identified which allowed for these compounds to be identified and distinguished without separation for the first time. Copyright © 2012 John Wiley & Sons, Ltd.