Psilocybe zapotecorum, a blue-bruising mushroom used ceremonially by indigenous groups in southeastern Mexico, was characterized through morphology, cultivation, chemical profiling, and full genome sequencing. The genome revealed the architecture of the psilocybin gene cluster, serving as a reference for Psilocybe clade I. Chemical analysis showed a psilocybin concentration of 17.9 ± 1.7 mg/g (range 10.6–25.7 mg/g, n = 7) and lesser amounts of related tryptamines, yielding a combined tryptamine concentration of 22.5 ± 3.2 mg/g. These findings indicate P. zapotecorum is a potent and chemically variable species. Cultivation and genetic analysis help demystify these mushrooms, expanding understanding beyond the molecule as clinical psilocybin studies advance.
*Psilocybe zapotecorum*, a mushroom with historical indigenous use, contains a potent psilocybin concentration of 17.9 mg/g, ranging from 10.6-25.7 mg/g across seven samples. Its full genome, revealing the psilocybin gene cluster, offers new biological insights. Combining Fungal Biology and Applications with Chemical synthesis and alkaloids, the work profiles its complex tryptamine chemistry, including psilocin and baeocystin. A total tryptamine concentration reached 22.5 mg/g. These findings advance Psychedelics and Drug Studies by illuminating the diversity of naturally occurring psilocybin sources.