The diversity of plant specialized metabolites has been extensively studied to better understand plant interactions with and responses to the environment, insects/vertebrates, and other plants. Pilocarpus, a Neotropical genus in Rutaceae (Citrus Family) most notably known as the main source of pilocarpine for the treatment of glaucoma, is a chemically diverse genus rich in alkaloids, terpenoids and coumarins. An overharvesting of Pilocarpus microphyllus has led to near extinction of natural populations of this species; therefore, alternative sources of pilocarpine in other species, as well as an understanding of the important factors associated with increases in compound yields would greatly help reduce this destruction and maintain diversity. The main aim of this research was to elucidate how phylogenetic relationships and adaptation to environmental variation shape phytochemical diversity in Pilocarpus. Phylogenetic regression models determined that phylogeny was better at predicting chemical traits when compared to models that only included certain bioclimatic factors. In addition, ecological niche models were used to determine the bioclimatic factors that contribute most to species distribution, including the identification of a large probability for niche overlap between species. Ancestral niche reconstruction identified geographically defined clades, as well as a potential tropical origin for the Pilocarpus clade. Next, an ecometabolomic analysis of P. pennatifolius revealed environmental variables correlated with alkaloid and phenolic variation. Finally, a population study on P. pennatifolius, P. spicatus, and P. riedelianus discovered significant differences in the genetic diversity and structure of these wild populations. Studies utilizing both environmental and phylogenetic factors are essential to tease out the intricate processes in the evolution of chemical diversity in plants. These methods can benefit fields such as conservation management, ecology, and evolutionary biology.