Abstract: To investigate the effect of substitution sites of the nitro group on the UV photodissociation (UVPD) of nitrobenzimidazole isomers, UVPD mass spectra and spectra of protonated ions of 2-nitrobenzimidazole （2-NBI）, 5-nitrobenzimidazole (5-NBI) and 7-nitrobenzimidazole (7-NBI) were studied by a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer combined with a tunable UV laser. Experimental results showed that the method of UVPD can distinguish the three isomers effectively. Although the UVPD spectrum of 2-NBI extended to about 360 nm, the UVPD spectra of 5-NBI and 7-NBI ended at 290 and 300 nm, respectively. As to UVPD mass spectra, the dissociation products were more diverse than the ones observed in CID experiments. And the yields of some ions significantly depended on the wavelength applied. Combined with theoretical calculations, the most important dissociation channels for the three ions were investigated in detail. For 2-NBI, two fragment ions of m/z 147 and 146 were observed, which were formed by the loss of OH· and H₂O resulting from one and two H atom transfers on the N atom of imidazole ring, respectively. For 5-NBI, the process of H transfer couldn’t happen due to the long distance between the nitro group and the imidazole ring. Therefore, only fragment ions of m/z 148 by the loss of O atom were observed. For 7-NBI, its nitro group located between the benzene ring and the imidazole ring, thus only one H atom could be transferred. Therefore, only the m/z 147 (OH· loss) fragment ions could be formed in the process. The channels for the generation of fragment ions of m/z 134 and 106 were also analyzed and discussed in this paper.