Clusia is a remarkable genus of Neotropical woody plants as its members engage in either C3 photosynthesis or employ, to varying degrees, crassulacean acid metabolism (CAM) photosynthesis. Information about the evolutionary history of CAM in Clusia is scarce. Restriction site-associated sequencing of 64 species (20% of the genus) provided strong support for most of the previously recognized nine lineages. Ancestral reconstruction using maximum parsimony or maximum likelihood under a one-rate model suggested that CAM evolved at least four times independently from a most recent common ancestor (MRCA) with C3, whereas a maximum likelihood two-rate model suggested that CAM was already present in the MRCA followed by reversions to C3 in several lineages. Phylogenetic generalized least square analysis assessed variation in seven leaf anatomical traits and CAM activity measured as δ 13C. Results indicate that CAM is highly correlated with palisade mesophyll layer thickness and cell size. In addition, correlation between 19 bioclimatic variables and δ 13C was evaluated. It was found that CAM is positively correlated with habitats with a more severe dry season and greater precipitation seasonality. Since CAM is weakly and/or only periodically expressed in many Clusia spp., and thus not readily reflected in δ 13C, future analysis of phylogenetically-informed CAM expression in Clusia must include physiological measurements such as CO2 exchange and/or diel changes in leaf acidity for each species under investigation.