A comparative study on the phenotypic and genetic characteristics among Acidithiobacillus ferrooxidans (AF2), a typic strain ATCC23270 and a previously isolated strain AF3 was performed. AF2 can use ferrous ion (Fe2+) or elemental sulfur (S0) as sole energy source, but oxidizes S0 more effectively than Fe2+, which is different from ATCC23270 and AF3. The G+C content of AF2 is 51.8% (molar fraction), however, ATCC23270 and AF3 strains have G+C content of 63.7% and 64.8% (molar fraction), respectively. The DNA-DNA hybridization results show that AF2 has 41.53% and 52.38% genome similarity to ATCC 23270 and AF3, respectively, but AF3 has a high genome similarity of 89.86% to ATCC 23270 strain. Rusticyanin (rus) and subunit Ⅲ of aa3-type cytochrome oxidase (coxC) genes are not detected in AF2, but Fe2+ oxidase (iro) gene can be detected. To understand the genomic organization of iro gene, a cosmid library of AF2 genome was constructed and iro gene-containing clone was screened. The sequencing result shows that although the nucleotide sequence of iro gene in AF2 is completely identical to that of ATCC 23270 strain, its genomic organization is different from that of ATCC 23270. In AF2, iro is located at downstream of pur A gene, while it is located at downstream of petC-2 gene in ATCC 23270 strain. These results indicate that AF2 is a novel strain of A. ferrooxidans, and that phenotypic differences among the strains of A. ferrooxidans are closely correlated with their genetic polymorphisms.