Abstract:Chlorella is one of the most important economically unicellular green algae, but its hundreds of varieties make it difficult to be distinguished based only on the morphological, physiological and biochemical features. To analyze the interspecies and intraspecies relationships and genetic variation of Chlorella species, two kinds of molecular markers were applied. One was the internal transcribed spacer (ITS) of nuclear rDNA and the other was the chloroplast rbcL gene encoding ribulose1,5bisphosphate carboxylase/oxygenase large subunit. The 6 〖WTBX〗Chlorella〖WTB1〗 strains studied included C. pyrenoidosa F5, F9, 820, C. protothecoides F2, C. vulgaris Cvq and C. ellipsoidea Ce. In addition to sequences retrieved from GenBank, a total 15 ITS and 15 rbcL sequences were carried out by sequence comparison, genetic distance calculation and phylogenetic tree construction, respectively. Results showed that the length of ITS region was almost the same among species intrasections, but the length of ITS1 and ITS2 varied significantly between species. However, the length of rbcL sequence was always conservative at inter and intraspecies levels. The genetic distances of 15 ITS sequences ranged from 0.000 to 0.663, and those of 15 rbcL sequences varied from 0.000 to 0.216. The phylogenetic relationships among the 6 Chlorella strains drawn by ITS and rbcL were as follows: First, C. protothecoides F2 showed very close relationship with C. pyrenoidosa F5 and F9. Second, C. pyrenoidosa 820 clustered closely with C. vulgaris Cvq. Third, C. ellipsoidea Ce showed the farthest distance with the other five Chlorella strains. This paper indicated that phylogenetic analysis and molecular identification of the genus Chlorella could be validated based on the combination of the highly variable ITS sequence and the relatively conservative chloroplast rbcL gene.