Modern hot springs provide good analogs of early Earth geothermal systems where microbial metal(loid) resistance genes probably first evolved. Arsenic (As) is a metal(loid) with a range of inorganic and organic speciations, dependent on environmental geochemical conditions. The evolution of genes encoding for arsenic resistance and energy conservation almost certainly reflects As speciation in ancient geothermal systems, many of which were likely to have been anoxic before the rise of atmospheric oxygen. We investigated the quantitative chemical speciation of arsenic, in conjunction with a metagenomic analysis of the extremophile microbiome, in Champagne Pool, New Zealand (Wai-o-tapu, North Island). In Champagne Pool, arsenite (As[III]O33-) constituted the major As species in the pool, rim and outflow channel; however, various thioarsenate species were also ubiquitously present in these sites as well as on the flood terrrace. In the outflow channel, the only instance of methylated arsenate was detected in waters flowing through a streamer-like biofilm. Metagenomic analyses revealed genes encoding for arsenate reductase at all sites, supporting an early evolution for arsenate resistance in even anoxic environments. Absence of the arsenite oxidase gene at all sites, however, suggests that arsenite detoxification (efflux pumping) evolved before energy conservation (electron transfer) from As(III) was possible. The detection of methylated arsenic in the outflow channel coincided with a major shift in relative abundance from Bacteria to Archaea, and possibly indicates a role for methylation in the arsenic resistance strategies of thermophilic Archaea. Our study highlights the importance of understanding metagenomic data in the context of biotic and abiotic factors influencing arsenic cycling, and yields clues for deciphering the evolution of microbial arsenic resistance genes.