BackgroundThe genusAcidithiobacillushas been widely concerned due to its superior survival and oxidation ability in acid mine drainage (AMD). However, the contribution of insertion sequence (IS) to their biological evolution and environmental adaptation is very limited. ISs are the simplest kinds of mobile genetic elements (MGEs), capable of interrupting genes, operons, or regulating the expression of genes through transposition activity. ISs could be classified into different families with their own members, possessing different copies.ResultsIn this study, the distribution and evolution of ISs, as well as the functions of the genes around ISs in 36Acidithiobacillusgenomes, were analyzed. The results showed that 248 members belonging to 23 IS families with a total of 10,652 copies were identified within the target genomes. The IS families and copy numbers among each species were significantly different, indicating that the IS distribution ofAcidithiobacilluswere not even.A. ferrooxidanshad 166 IS members, which may develop more gene transposition strategies compared with otherAcidithiobacillusspp. What’s more,A. thiooxidansharbored the most IS copies, suggesting that their ISs were the most active and more likely to transpose. The ISs clustered in the phylogenetic tree approximately according to the family, which were mostly different from the evolutionary trends of their host genomes. Thus, it was suggested that the recent activity of ISs ofAcidithiobacilluswas not only determined by their genetic characteristics, but related with the environmental pressure. In addition, many ISs especially Tn3 and IS110 families were inserted around the regions whose functions were As/Hg/Cu/Co/Zn/Cd translocation and sulfur oxidation, implying that ISs could improve the adaptive capacities ofAcidithiobacillusto the extremely acidic environment by enhancing their resistance to heavy metals and utilization of sulfur.ConclusionsThis study provided the genomic evidence for the contribution of IS to evolution and adaptation ofAcidithiobacillus, opening novel sights into the genome plasticity of those acidophiles.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09372-8.