Abstract Background The CO₂-concentrating mechanism associated to Crassulacean acid metabolism (CAM) alters the catalytic context for Rubisco by increasing CO₂ availability and provides an advantage in particular ecological conditions. We hypothesized about the existence of molecular changes linked to these particular adaptations in CAM Rubisco. We investigated molecular evolution of the Rubisco large (L-) subunit in 78 orchids and 144 bromeliads with C₃ and CAM photosynthetic pathways. The sequence analyses were complemented with measurements of Rubisco kinetics in some species with contrasting photosynthetic mechanism and differing in the L-subunit sequence. Results We identified potential positively selected sites and residues with signatures of co-adaptation. The implementation of a decision tree model related Rubisco specific variable sites to the leaf carbon isotopic composition of the species. Differences in the Rubisco catalytic traits found among C₃ orchids and between strong CAM and C₃ bromeliads suggested Rubisco had evolved in response to differing CO₂ concentration. Conclusions The results revealed that the variability in the Rubisco L-subunit sequence in orchids and bromeliads is composed of coevolving sites under potential positive adaptive signal. The sequence variability was related to δ¹³C in orchids and bromeliads, however it could not be linked to the variability found in the kinetic properties of the studied species.