Based upon five contigs coding for carbonic anhydrase(CA)from a pyrosequencing transcriptome of Myrmecia incisa, gene-specific primers were designed for CA gene cloning from this green microalga. Five CA genes, named MiαCA1, MiαCA2, MiβCA1, MiβCA2 and Mi_γCA were cloned by using the technique of rapid amplification of cDNA ends. The open reading frames of the cloned full-length cDNAs were 963 bp, 1 089 bp, 1 041 bp, 738 bp and 687 bp in length, encoding putative proteins composed of 320, 362, 346, 245 and 228 amino acids, respectively. The encoded CA proteins were rich in hydrophobic amino acids which accounted for 41. 25%, 45. 31%, 43. 35%, 42. 45% and 43. 42% of total amino acids, respectively. Neighbor-joining(NJ)phylogenetic tree inferred from the putative proteins of CA genes indicated that these cloned CA genes were divided into three clades: MiαCA1 and MiαCA2 in α-CA clade, MiβCA1 and MiβCA2 in β-CA one and Mi_γCA in γ-CA one. MiαCA1 and MiαCA2 are characterized by 3 conserved His residues, MiβCA1 and MiβCA2 are by 2 conserved Cys and 1 conserved His residues whereas Mi_γCA is found to use Arg, His and Asn instead of the reported 3 residues of His to bind Zn²⁺ to form their catalysis centre. MiαCA1, clustered with CAH3 from Chlamydomonas reinhardtii in the NJ phylogenetic tree, had a two-part leader sequence, suggesting that it might be localized and associated with PSII in the thylakoid lumen like CAH3. MiαCA2, close to CAH1 after protein alignment, had a signal peptide which could lead the mature MiαCA2 to the periplasmic space resembling CAH1 in C. reinhardtii. It was predicted that this CA could function as CAH1 to transfer HCO₃^- into CO₂ for photosynthesis. Two β-CAs, MiβCA1 and MiβCA2, were clustered with CAH7 and CAH 8 from C. reinhardtii, which might be involved in interconversion between HCO₃^- and CO₂ in the cytoplasm of M. incisa. Mi_γCA, grouped with γ-CA from higher plants, was expected to be associated with the complex I of mitochondrial electron transport chain to work. The 5 cloned CA genes from M. incisa should be localized at various regions of the algal cells to cooperate with each other for the reversible interconversion of CO₂ and HCO₃^- so as to modify pH and transfer CO₂ among the organelles.