Abstract: Synthetic cathinones are a class of new psychoactive substances, characterized by strong addictiveness, great diversity, rapid updates, and similar structures. Among them, 3-fluoromethcathinone (3-FMC) and 4-fluoromethcathinone (4-FMC) are two positional isomers of the second-generation synthetic cathinone, fluoromethcathinone. Their structures are very similar, therefore, the accurate qualitative analysis of these isomers remains a challenge in current judicial appraisals. In this study, the fragmentation patterns of positional isomers 3-FMC and 4-FMC were investigated by gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOF MS) and liquid chromatography-Q-Orbitrap mass spectrometry (LC-Q-Orbitrap/MS). Then, the mass differences of 3-FMC and 4-FMC fragments were further analyzed by the relationship between chemical structures and fragment ion abundances. Computational studies further demonstrated the experimental results using Gibbs free energy profiles and electronic properties of possible fragments. In addition, the fragmentation mechanisms of 3-FMC and 4-FMC were investigated by applying density functional theory calculation, which can more accurately elucidate the mass spectral behavior and structure characteristics of compounds as reflected by mass spectrometric data. The results showed that the side chain of the benzene ring in 3-FMC is prone to break and generate a fragment ion at m/z 58. It was found that the electron cloud densities at the ortho and para positions of the benzene ring change significantly due to the strong electron-withdrawing inductive effect of the fluorine atom. This, in turn, leads to different fragmentation patterns of the benzene ring side chains of different positional isomers. Specifically, the side chain on the benzene ring of 3-FMC is prone to heterolytic cleavage to lose one molecule of CH₄ and generate a fragment ion with m/z 148. In contrast, the side chain on the benzene ring of 4-FMC tends to homolytic cleavage to lose one molecule of CH₃· and generate a fragment ion at m/z 149. Structures of possible fragments were screened based on relative Gibbs free energy to confirm the most stable isomer of fragment ions. The first stage of fragmentation with one H₂O loss is a variant of α-hydroxy N-phenylimines rearrangement reaction, which consists of three steps of σ-migration and acts as the rate-determinant step of the fragmentation process to form the four-membered ring side chain fragment (m/z 164, IM6). Further theoretical prediction was conducted using relative Gibbs free energy. All possible isomers that retain the fluorophenyl structure are screened to determine the most stable structure. The results of the fragmentation patterns obtained from computational chemistry are consistent with the experimental results of mass spectrometry analysis. The study provides an important reference and a basis for the accurate identification of the fluoromethcathinone isomers 3-FMC and 4-FMC.