Abstract: Fentanyl is a synthetic opioid with strong anesthetic and analgesic properties. A significant challenge for the legislative control of fentanyl abuse is the ever-growing list of emerging derivatives. New fentanyl analogues have been constantly synthesized by alterations to the fentanyl structure to circumvent regulatory oversight and avoid laboratory scrutiny. Therefore, the reliable identification of fentanyl analogues is of great significance for public security. In this study, the tandem mass spectrometry (MS/MS) data for 46 fentanyl analogues were acquired using dielectric barrier discharge ionization mass spectrometry (DBDI-MS), and their fragmentation pathways were further investigated. The fentanyl analogues were subjected to the ionization of both DBDI and electrospray ionization (ESI). Despite a lower overall ion intensity, DBDI produced more characteristic fragment ions, providing comprehensive structural information for the identification of fentanyl analogues. Abundant protonated molecules were readily observed for fentanyl analogues in the positive ionization mode. The protonated fentanyl compounds were chosen as precursor ions for subsequent collision-induced dissociation (CID). Structurally similar compounds generally exhibit consistent fragmentation behavior during the MS/MS analysis. Characteristic fragment ions at m/z 105 and m/z 188 were observed for two important fentanyl precursors (N-phenethyl-4-piperidone and 4-anilino-N-phenethylpiperidine). When structural substitution occurred on the phenethyl group or piperidine ring of fentanyl, a neutral loss of 149 u could be found, indicating the cleavage of the C—N bond at the 4′ position of piperidine ring and the generation of N-phenylpropionamide. On the other hand, the substitution at the N-propionyl or N-phenyl group led to the formation of a fragment ion at m/z 188, corresponding to N-phenethyl-4-piperidine cation. Therefore, the 46 fentanyl analogues investigated were categorized into two groups according to their fragmentation patterns. Moreover, a rapid method was developed for the analysis of fentanyl in powder samples using DBDI-MS. Good linearity was obtained in the concentration range of 1-20 mg/kg (r=0.999 3). The limit of detection (LOD) and quantification (LOQ) for fentanyl were 0.3 and 1 mg/kg, respectively, with the relative standard deviations (RSDs) less than 2.6%. The current study demonstrates a promising potential for reliable screening of fentanyl analogues with enhanced operation simplicity, analysis speed, and detection throughput.