Differentiating halogen positional isomers of aromatic compounds using gas chromatography and mass spectrometry is a challenging task due to insufficient chromatographic separation and close similarity in the mass spectra of the isomers. For synthetic cannabinoids (SCs), a growing number of halogen derivatives have emerged, while there is no convenient and easily accessible procedure to differentiate the regioisomers of illegal drugs. In the current study, FUB-JWH-018 and its five isomers having structural and regioisomeric features were synthesized to investigate mass spectrometric differentiation by gas chromatography-electron ionization-tandem mass spectrometry (GC-EI-MS-MS). The ions at m/z 379, 378, and 362 were selected as precursor ions for this study based on their EI mass spectra. Differences in relative intensity of product ions were observed among the isomers, enabling feasible regioisomeric differentiation by mass spectrometry. Furthermore, high reproducibility of the product ion spectra at the optimized collision energy was confirmed and FUB-JWH-018 was successfully identified from illegal drug market products, demonstrating the reliability and practicality of the method. The characteristic properties in fragmentation with the mechanistic pathways are also described. This is the first report on mass spectrometric differentiation of the isomers of SCs bearing substituted regioisomeric fluorobenzyl moiety on 1H-indole core by GC-EI-MS-MS. The procedure has great potential to help in differentiating halogen positional isomers, providing clues to discriminate newly encountered designer drugs in the fields of analytical chemistry and forensic toxicology. (C) 2017 Elsevier B.V. All rights reserved.