Members　of　the　genus　Acidithiobacillus,　which　can　adapt　to　extremely　high　concentrations　of　heavy　metals,　are　universally　found　at　acid　mine　drainage　(AMD)　sites.　Here,　we　performed　a　comparative　genomic　analysis　of　37　strains　within　the　genus　Acidithiobacillus　to　answer　the　untouched　questions　as　to　the　mechanisms　and　the　evolutionary　history　of　metal　resistance　genes　in　Acidithiobacillus　spp.　The　results　showed　that　the　evolutionary　history　of　metal　resistance　genes　in　Acidithiobacillus　spp.　involved　a　combination　of　gene　gains　and　losses,　horizontal　gene　transfer　(HGT),　and　gene　duplication.　Phylogenetic　analyses　revealed　that　metal　resistance　genes　in　Acidithiobacillus　spp.　were　acquired　by　early　HGT　events　from　species　that　shared　habitats　with　Acidithiobacillus　spp.,　such　as　Acidihalobacter,　Thiobacillus,　Acidiferrobacter,　and　Thiomonas　species.　Multicopper　oxidase　genes　involved　in　copper　detoxification　were　lost　in　iron-oxidizing　Acidithiobacillus　ferridurans,　Acidithiobacillus　ferrivorans,　and　Acidithiobacillus　ferrooxidans　and　were　replaced　by　rusticyanin　genes　during　evolution.　In　addition,　widespread　purifying　selection　and　the　predicted　high　expression　levels　emphasized　the　indispensable　roles　of　metal　resistance　genes　in　the　ability　of　Acidithiobacillus　spp.　to　adapt　to　harsh　environments.　Altogether,　the　results　suggested　that　Acidithiobacillus　spp.　recruited　and　consolidated　additional　novel　functionalities　during　the　adaption　to　challenging　environments　via　HGT,　gene　duplication,　and　purifying　selection.　This　study　sheds　light　on　the　distribution,　organization,　functionality,　and　complex　evolutionary　history　of　metal　resistance　genes　in　Acidithiobacillus　spp.　IMPORTANCE　Horizontal　gene　transfer　(HGT),　natural　selection,　and　gene　duplication　are　three　main　engines　that　drive　the　adaptive　evolution　of　microbial　genomes.　Previous　studies　indicated　that　HGT　was　a　main　adaptive　mechanism　in　acidophiles　to　cope　with　heavy-metal-rich　environments.　However,　evidences　of　HGT　in　Acidithiobacillus　species　in　response　to　challenging　metal-rich　environments　and　the　mechanisms　addressing　how　metal　resistance　genes　originated　and　evolved　in　Acidithiobacillus　are　still　lacking.　The　findings　of　this　study　revealed　a　fascinating　phenomenon　of　putative　cross-phylum　HGT,　suggesting　that　Acidithiobacillus　spp.　recruited　and　consolidated　additional　novel　functionalities　during　the　adaption　to　challenging　environments　via　HGT,　gene　duplication,　and　purifying　selection.　Altogether,　the　insights　gained　in　this　study　have　improved　our　understanding　of　the　metal　resistance　strategies　of　Acidithiobacillus　spp.