We characterized the diversity of sulphate-reducing bacteria (SRB) associated with South African gold mine boreholes and deep aquifer systems in Washington State, USA. Sterile cartridges filled with crushed country rock were installed on two hydrologically isolated and chemically distinct sites at depths of 3.2 and 2.7 km below the land surface (kmbls) to allow development of biofilms. Enrichments of sulphate-reducing chemolithotrophic (H2) and organotrophic (lactate) bacteria were established from each site under both meso- and thermophilic conditions. Dissimilatory sulphite reductase (Dsr) and 16S ribosomal RNA (rRNA) genes amplified from DNA extracted from the cartridges were most closely related to the Gram-positive species Desulfotomaculum thermosapovorans and Desulfotomaculum geothermicum, or affiliated with a novel deeply branching clade. The dsr sequences recovered from the Washington State deep aquifer systems affiliated closely with the South African sequences, suggesting that Gram-positive sulphate-reducing bacteria are widely distributed in the deep subsurface.