The isomers of fluoro-butyrylfentanyl, fluoro-isobutyrylfentanyl, and fluoro-methoxyacetylfentanyl, in which the position of fluorine on the N -phenyl ring varies, were synthesized, characterized, and differentiated by infrared (IR) spectroscopy, liquid chromatography/mass spectrometry (LC/MS), and gas chromatography/mass spectrometry (GC/MS). The isomers could be clearly differentiated by their IR spectra. In the LC/MS chromatograms, the separation of the fluoro-butyrylfentanyl and fluoro-isobutyrylfentanyl isomers was insufficient. However, in the GC/MS extracted ion chromatograms, all compounds were completely separated. The LC/MS and GC/MS mass spectra of the isomers were similar, demonstrating that it is difficult to distinguish the positional isomers of fluorinated fentanyl analogs by their mass spectra.