The chromosomal DNA of Thiobacillus thiooxidans and T. versutus was digested with restriction enzymes, blotted to nylon membrane by the way of Southern, and hybridized with the gene probe of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) from T. ferrooxidans. The result showed that T. thiooxidans exhibited high homology with the probe anc T. versutus was less homology with it. However T. versutus can hybridize with the gene probe of RubisCO from Rhodobacter sphaeroides, which indicated that they had high homology with each other. As RubisCO was highly reserved in the evolutionary, they should be divided into different group.