Abstract: Accurate determination of 2,4-diaminotoluene is of great significance due to its carcinogenic effect and its common existence in food packaging materials. However, 2,4-diaminotoluene shares similar EI-MS behavior with its isomers, and it is difficult to separate 2,4-diaminoto-luene with its isomers on many common chromatographic column. Thus, it is a great challenge for differentiation of carcinogenic 2,4-diaminotoluene with its isomers. In this work, imidization of 5 commercial available diaminotoluenes with benzaldehyde was carried out, and the products were analyzed by gas chromatography-mass spectrometry. Both of the two nonadjacent amino groups in m- or p-diaminotoluene underwent imidization with benzaldehyde, respectively, while derivatization of o-diaminotoluene resulted in 1-benzyl-2-phenyl-methyl-1H-benzod imidazole and 2-phenyl-methyl-1H-benzod imidazole at room temperature, while only led to 2-phenyl-methyl-1H-benzod imidazole when heated. All of the derivatives were easily separated on a common HP-5MS capillary column. They underwent the loss of H atom or phenyl radical in EI-MS. Due to the ortho-effect, from 2,3-diaminotoluene underwent dissociation to give a characteristic product ion at m/z 207, and thus they can be differentiated from 3,4-diaminotoluene. Both of the imino groups in the derivative of 2,4-diaminotoluene participated in the fragmentation reaction, which resulted in characteristic product ions at m/z 207 and m/z 195. Both of the imino groups were in the conjugated position in the derivative of 2,5-diaminotoluene, and thus the molecular ion was especially stable with much high abundance. Thus, all of these derivatives were recognized by EI-MS. Theoretical calculations at the B3LYP/6-31G(d) level were carried out to probe the EI-MS fragmentation of the imidization product of the 2,3-diaminotoluene, in which migration of one of the methyl H atoms to the neighboring N atom led to the loss of the benzyl radical. The results provide a promising solution for simultaneous determination of diaminotoluene isomers.