The gold and uranium mines of the Free State gold fields in South Africa are no longer mined underground but provide a rich source of biological data for analysis from the different rehabilitation programmes designed to revegetate the above-ground tailings. Some of the programmes have incorporated the application of mycorrhizal fungal inoculum and the aim of this study was to assess the arbuscular mycorrhiza (AM) fungal diversity in the roots of trees being used for two different phytoremediation trials. The trees sampled were indigenous acacias (Vachellia and Senegalia spp.) and two tailings assessed were (i), planted with trees which had been inoculated with crude AM fungal inocula in the nursery and received no organic inputs and (ii), ameliorated with garden refuse, on which the acacias were natural colonisers. The approach was to target and amplify the small subunit rRNA gene sequences, a marker gene widely used in ecological studies for phylogenetic analysis to determine AM fungal diversity. This study identified 17 different AM fungal species that fell within 8 genera, namely, Diversispora, Rhizophagus, Scutellospora, Claroideoglomus, Cetraspora, Sclerocystis, Glomus and Redecker. From the planted mine tailing 8 genera and 13 species were identified of which 5 species were unique to the tailing, and from the garden refuse-ameliorated mine tailing 6 genera and 12 species were identified of which 4 species were unique to the tailing. A limited soil physico-chemical analysis of the substrata revealed no significant difference between the sites except for the element Al, whose concentration on site (i) was almost double that of site (ii). The elements Pb, Hg, Mo, Ni, Pt and Th were not detected. The results represent the first AM fungal diversity study from South Africa sampling root DNA and report 9 first records of AM fungal species in South Africa. A high number of AM fungal taxa were identified when compared with other diversity studies on metal-contaminated sites, all of which exhibited substantially higher levels of pollutant elements than the Free State sites.