Abstract Ribulose-l, 5-bisphosphate carboxylase/oxygenase (Rubisco) is a bifunctional enzyme in plants and it can be instrumental in the continual adaption to the variations in the concentrations of CO2. The large subunit of Rubisco is encoded by choloplast rbcL gene. In the current analysis, based on 59 sequences from Brassicaceae species and the evolutionary analyses from the site-specific model, the 289th codon site was found to be under positive selection in rbcL gene. The results demonstrated that positive selection has played a major role in the adaptive evolution of rbcL. The site was in the α/β-barrel active center located on the C-terminal domain of the large subunit of Rubisco and the amino acid found here was isoleucine (Ile) or valine (Val). The changes of this domain may be associated with the adaptive evolution of this protein to the various habitats for Brassicaceae species.