Acidithiobacillus ferrooxidansserves as a model chemolithoautotrophic organism in extremely acidic environments, which has attracted much attention due to its unique metabolism and strong adaptability. However, little was known about the divergences along the evolutionary process based on whole genomes. Herein, we isolated six strains ofA. ferrooxidansfrom mining areas in China and Zambia, and used comparative genomics to investigate the intra-species divergences. The results indicated thatA. ferrooxidansdiverged into three groups from a common ancestor, and the pan-genome is ‘open’. The ancestral reconstruction ofA. ferrooxidansindicated that genome sizes experienced a trend of increase in the very earliest days before a decreasing tendency during the evolutionary process, suggesting that both gene gain and gene loss played crucial roles inA. ferrooxidansgenome flexibility. Meanwhile, 23 single-copy orthologous groups (OGs) were under positive selection. The differences of rusticyanin (Rus) sequences (the key protein in the iron oxidation pathway) and type IV secretion system (T4SS) composition in theA. ferrooxidanswere both related to their group divergences, which contributed to their intraspecific diversity. This study improved our understanding of the divergent evolution and environmental adaptation ofA. ferrooxidansat the genome level in extreme conditions, which provided theoretical support for the survival mechanism of living creatures at the extreme.