5-bisphosphate carboxylase/oxygenase is one of the key enzymes catalyzing the CO₂ fixation in the Calvin cycle. RbcSplays a role in regulating the Rubisco activity. In this studyChlorel a pyrenoidosa strains F-9 and 820 were cloned and analyzedrespectively. Two cDNA sequences of 245 bp were obtained according to a pair of degenerate primers cDNA sequences were cloned by rapid amplification of cDNA ends technology. The full-length F-9 was 861 bp including 66 bp 5′-untranslated regionrbcS sequence of 820 was consisted of 68 bp 5′-untranslated region284 bp 3′-untranslated region and 555 bp ORF. The F-9 ORF contained 41 deduced amino acids transit peptide and 140 amino acids mature peptide. The transit peptide of 820 ORF 44 amino acidsand the mature peptide was the same length as that of F-9. Structural analysis revealed one Kozak consensus sequence around the start codon ATGand a 5 bp TGTAA tailing signal in the 3′-untranslated region. The similar conserved sequence of YYDGRYWTMWKLPMFG in higher plants were also found in the two cDNAs with one or two amino acids differences in the second and fourth positionrespectively. Similarity analysis showed that the highest identity C. pyrenoidosa and F-9 showed 64% identity with 820 genes shared wide and moderate similarities with other green algae and high plants. This study provided materials for further research on the function and expression of genes.