The family Acidiferrobacteraceae (order Acidiferrobacterales) currently contains three genera of chemolithoautotrophs: Sulfuricaulis (2016), Sulfurifustis (2015) and Acidiferrobacter (2011). While the two former are neutrophilic sulfur oxidizers isolated from lake sediments in Japan, the latter is an extremely acidophilic, moderately osmophilic, thermotolerant iron/sulfur oxidizer known to occur in macroscopic streamers in Rio Tinto, Spain and in acid waters worldwide. The type strains of both Sulfuricaulis limnicola (HA5T) and Sulfurifustis variabilis (skN76T) have been sequenced, and the draft genome of the ZJ isolate of Acidiferrobacter thiooxydans (MDCF01) has recently been deposited in public databases. Despite this fact, little evidence on the genomic diversity and evolution of this group has been presented so far. Using comparative genomic analyses and phylogenetic reconstruction strategies, we explored the evolutionary information contained in the available genome sequences to shed light on the taxonomic status of a novel isolate of the genus Acidiferrobacter (SP-III/3; DSM 27195).