Rubisco activase is an ATP-dependent chaperone that facilitates dissociation of inhibitory sugar phosphates from the catalytic sites of ribulose-1,5-bisphosphate carboxylase/oxygenase during photosynthesis. In Arabidopsis, Rubisco activase is negatively regulated by dark-dependent phosphorylation of threonine 78. The prevalence of threonine 78 in Rubisco activase was investigated across sequences from 91 plant species, finding 29 (∼32%) species shared a threonine in the same position. Analysis of seven C3 species with an antibody raised against a threonine 78 phospho-peptide demonstrated that this position is phosphorylated in multiple genera. However, light-dependent dephosphorylation of threonine 78 was observed only in Arabidopsis. Further, phosphorylation of threonine 78 could not be detected in any of the four C4 grass species examined. The results suggest that despite conservation of threonine 78 in Rubisco activase from a wide range of species, a regulatory role for phosphorylation at this site is more limited. This provides a case study for how variation in post-translational regulation can amplify functional divergence across the phylogeny of plants beyond what is explained by sequence variation in a metabolically important protein.