C-4 photosynthesis allows increased photosynthetic efficiency because carbon dioxide (CO2) is concentrated around the key enzyme RuBisCO. Leaves of C-4 plants exhibit modified biochemistry, cell biology, and leaf development, but despite this complexity, C-4 photosynthesis has evolved independently in at least 45 lineages of plants. We found that two independent lineages of C-4 plant, whose last common ancestor predates the divergence of monocotyledons and dicotyledons about 180 million years ago, show conserved mechanisms controlling the expression of genes important for release of CO2 around RuBisCO in bundle sheath (BS) cells. Orthologous genes from monocotyledonous and dicotyledonous C-3 species also contained conserved regulatory elements that conferred BS specificity when placed into C-4 species. We conclude that these conserved functional genetic elements likely facilitated the repeated evolution of C-4 photosynthesis.