The low‐energy collision‐induced dissociation (CID) of the carboxylate anions generated by fast atom bombardment ionization of monohydroxy unsaturated fatty acids derived from oleic, linoleic, linolenic and arachidonic acids were studied in a tandem quadrupole mass spectrometer. The collisional activation spectra revealed structurally informative ions as to the position of the hydroxyl substituent in relationship to the sites of unsaturation. Five mechanisms are proposed for the fragmentation of hydroxy substituted unsaturated fatty acids and are dependent upon the presence of α‐ or β‐unsaturation sites. These mechanisms include charge‐remote allylic fragmentation, charge‐remote vinylic fragmentation, charge‐driven allylic fragmentation, charge‐driven vinylic fragmentation, and homolytic fragmentation by an oxy‐Cope rearrangement process. The assignment of specific fragmentation pathways was supported in many instances with deuterium‐labeled analogs. Although no single fragmentation mechanism appears to predominate, a rational approach to the interpretation of these CID spectra is proposed. The CID spectra of unknown compounds could be used to establish the hydroxyl substituent position in relationship to certain sites of unsaturation but would not be indicative of all double bond locations. The oxy‐Cope rearrangement is specific for a structural unit, namely the 3‐hydroxy‐1,5‐diene moiety.