Using high-performance liquid chromatography coupled to high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC/ESI-Q-TOF-MS), we have developed a new method for detection and identification of furan fatty acids (F-acids), which are widely distributed in living organisms and foods as minor lipid components and are known to have antioxidant and anti-inflammatory effects. For this purpose, total fatty acids prepared from the testis lipids of Japanese chum salmon (Oncorhynchus keta) were examined without any concentration or isolation of F-acids. In negative ESI mode, F-acids gave a prominent [M−H]− ion, by which individual F-acids could be detected and identified. High-resolution extracted ion chromatograms clearly showed the occurrence of five major F-acid homologs as already reported by GC/MS. The method was successfully applied to several fish samples and revealed the occurrence of F-acids for the first time in the two New Zealand fish, hoki (Macruronus novaezelandiae) and school shark (Galeorhinus galeus).