Psilocin (4‐hydroxy‐N,N‐dimethyltryptamine, 4‐HO‐DMT) and bufotenine (5‐hydroxy‐N,N‐dimethyltryptamine, 5‐HO‐DMT), which are both naturally occurring compounds, are classified as controlled substances in numerous countries due to their pharmacological activities and recreational usage. There are two other benzene ring regioisomers, 6‐hydroxy‐N,N‐dimethyltryptamine (6‐HO‐DMT) and 7‐hydroxy‐N,N‐dimethyltryptamine (7‐HO‐DMT), which are not classified by name as controlled substances, and which were synthesized for this current work. The four isomers were analyzed using routine methodologies employed by the Israel's Police Division of Identification and Forensic Science (DIFS) Laboratory, namely thin layer chromatography (TLC), Fourier transform infrared spectroscopy (FTIR), and gas chromatography mass spectroscopy (GC‐MS). It was found possible to differentiate the four isomers. Forensic specimens that were suspected to be psilocybe mushrooms were examined, confirming that it is now possible to unequivocally identify the presence of psilocin and rule out the presence of its other isomers.