C₄ photosynthesis without Kranz anatomy (single-cell C₄ photosynthesis) occurs in only 0.003% of known species of C₄ flowering plants. To add insight into the evolution of C₄ photosynthesis, we studied the tribe Orcuttieae (Poaceae: Chloridoideae), which has species that can grow under both aquatic and terrestrial conditions, and utilize single-cell C₄ photosynthesis when growing submerged. Carbon isotope ratios from aquatic, floating, and terrestrial leaves were in the range −12.25 to −14.31, suggesting that all species carry out C₄ photosynthesis. Using light microscopy, we examined the anatomy of aquatic, floating and terrestrial leaves from eight of the nine species in the tribe to assess the pattern of evolution of C₄ photosynthesis and Kranz anatomy among these vernal pool grasses. Kranz anatomy was present in all floating and terrestrial leaves of Orcuttia californica, O. inaequalis, O. pilosa, O. tenuis, O. viscida,Tuctoria greenei, T. mucronata, and Neostapfia colusana. Although carbon isotope data indicated C₄ photosynthesis, aquatic leaves of all members of Orcuttia lacked Kranz anatomy, while aquatic leaves of Tuctoria and Neostapfia possessed Kranz anatomy. When considered in a phylogenetic context, these findings support previously proposed hypotheses suggesting that Orcuttieae are derived from a terrestrial ancestor and are now becoming more specialized to an aquatic environment.