C4 photosynthesis has evolved multiple times from ancestral C3 species. Carbonic anhydrase (CA) catalyzes the reversible hydration of CO2 and is involved in both C3 and C4 photosynthesis; however, its roles and the intercellular and intracellular locations of the majority of its activity differ between C3 and C4 plants. To understand the molecular changes underlying the evolution of the C4 pathway, three cDNAs encoding distinct β-CAs (CA1, CA2, and CA3) were isolated from the leaves of the C3 plant Flaveria pringlei. The phylogenetic relationship of the F. pringlei proteins with other embryophyte β-CAs was reconstructed. Gene expression and protein localization patterns showed that CA1 and CA3 demonstrate high expression in leaves and their products localize to the chloroplast, while CA2 expression is low in all organs examined and encodes a cytosolic enzyme. The roles of the F. pringlei enzymes were considered in light of these results, other angiosperm β-CAs, and Arabidopsis (Arabidopsis thaliana) "omics" data. All three F. pringlei CAs have orthologs in the closely related C4 plant Flaveria bidentis, and comparisons of ortholog sequences, expression patterns, and intracellular locations of their products indicated that CA1 and CA2 have maintained their ancestral role in C4 plants, whereas modifications to the C3 CA3 gene led to the evolution of the CA isoform that catalyzes the first step in the C4 photosynthetic pathway. These changes included the loss of the chloroplast transit peptide and an increase in gene expression, which resulted in the high levels of CA activity seen in the cytosol of C4 mesophyll cells.