Flavonoids exhibit a wide range of biological activities and the fragmentation characterization and differentiation of isomeric diglycosyl flavonoids is an important area of research. In this study, ultra-high-performance liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectrometry was used to identify two pairs of isomeric diglycosyl flavonoids: naringin/narirutin and neohesperidin/hesperidin. The two pairs of flavonoids were identified by product ion tandem mass spectra of the precursor ions [M-H]-. The results showed that characteristic product ions distinguishing flavonoids glycosylated with rhamnosyl-(alpha1 --> 2)-glucosyl from isomeric flavonoids glycosylated with rhamnosyl-(alpha1 --> 6)-glucosyl were the precursor ion [M-H]- and its characteristic product ions Y0- and [0.2X0-H]-.