The evolution of C4 photosynthesis from C3 ancestors eliminates ribulose bisphosphate carboxylation in the mesophyll (M) cell chloroplast while activating phosphoenolpyruvate (PEP) carboxylation in the cytosol. These changes may lead to fewer chloroplasts and different chloroplast positioning within M cells. To evaluate these possibilities, we compared chloroplast number, size and position in M cells of closely related C3, C3–C4 intermediate and C4 species from 12 lineages of C4 evolution. All C3 species had more chloroplasts per M cell area than their C4 relatives in high-light growth conditions. C3 species also had higher chloroplast coverage of the M cell periphery than C4 species, particularly opposite intercellular air spaces. In M cells from 10 of the 12 C4 lineages, a greater fraction of the chloroplast envelope was pulled away from the plasmalemma in the C4 species than their C3 relatives. C3–C4 intermediate species generally exhibited similar patterns as their C3 relatives. We interpret these results to reflect adaptive shifts that facilitate efficient C4 function by enhancing diffusive access to the site of primary carbon fixation in the cytosol. Fewer chloroplasts in C4 M cells would also reduce shading of the bundle sheath chloroplasts, which also generate energy required by C4 photosynthesis.