Abstract: Acidithiobacillus ferrooxidans (A.f) is a Gram-negative chemolithotrophic bacterium able to oxidize ferrous iron, elemental sulfur and inorganic sulfur compounds. Fe(II)-oxidizing enzyme plays key role in Fe(II)-oxidizing system. Different A.f strains, which were isolated and purified from various ecological niches, show obviously phenotypic diversity after comparison rates of growth and oxidation of Fe2+ . The Fe(II)-oxidizing enzyme gene(iro) for five strains was identified by PCR reactions uitilising primers and genomic DNA, then sequenced and multialignment. The results show there is a putative high variable region in CDS between the 187th and 195th bp and the sequence similarities are from 97% to 99%. For strain YTW, P187～189 Thr→Pro; For P193～195, Met→Asn for strain TK; Met→Ile for strain BY ; For P219 , all strains T→C. For upstream of CDS , sequences of all strains are identical to the Genebank sequence (E03451), but downstream of CDS, the sequences exist some variable sites too.