Psilocybe cubensis, a widely recognized psychoactive mushroom species, has played a significant role in both historical and modern therapeutic practices. This review explores the complex interplay between genetic diversity, strain variability and environmental factors that shape the biosynthesis of key psychoactive compounds, including psilocybin and psilocin. With many strains exhibiting substantial variability in their phenotypic characteristics and biochemical content, understanding and documenting this diversity is crucial for optimizing therapeutic applications. The review also highlights advances in cultivation techniques, such as submerged fermentation of the mycelium, and innovative analytical methodologies that have improved the precision of compound quantification and extraction. Although there is limited scientific information on P. cubensis due to nearly four decades of regulatory restrictions on psychedelic research, recent developments in genetic and biochemical studies are beginning to provide valuable insights into its therapeutic potential. Furthermore, this review emphasizes key knowledge gaps and offers insights into future research directions to advance the cultivation, scientific documentation of strain diversity, regulatory considerations and therapeutic use of P. cubensis.

Psilocybin-producing mushrooms exhibit considerable biochemical diversity, yet the extent of variability among strains within a single species under standardized conditions remains insufficiently characterized. In this study, we quantified psilocybin, psilocin, baeocystin, norbaeocystin, aeruginascin, and norpsilocin in the fruiting bodies of 14 distinct strains of Psilocybe cubensis. The mushroom strains were cultivated, dried, extracted, and analyzed under uniform laboratory conditions. Despite strict methodological standardization, total tryptamine concentrations varied by more than 7.8-fold among strains (from 2.62 to 20.65 mg/g), with psilocybin consistently emerging as the dominant compound. Analysis of individual fruiting bodies within the selected strains revealed substantial intra-strain variability, with coefficients of variation for psilocybin ranging from 12.81% to 23.39% between individual fruiting bodies. These findings demonstrate that both inter- and intra-strain biochemical heterogeneity persist even under controlled conditions, underscoring the challenges of standardizing whole mushroom preparations for research or therapeutic use. Our results highlight the importance of strain selection, rigorous chemical profiling, and dosing precision in future pharmacological and clinical applications of P. cubensis.