Culture-independent modes of analysis were chosen to investigate a microbial mat associated with the thermal waters of the Great Artesian Basin (GAB). 16S rDNA was amplified from total genomic mat DNA, and used to construct a clone library. Use of plasmid-specific primers to amplify the inserts from 92 selected recombinants proved to be an effective approach, and demonstrated that there were four size categories of insert: 1500 bp (62% of clones examined), 1400 bp (25% of clones examined), 500 bp (5% of clones examined) and 240 bp (8% of clones examined). Restriction enzyme analysis was evaluated for its ability to group the 1500 bp and 1400 bp size inserts into operational taxonomic units. Clone inserts were presumptively identified by analysis of partial sequence data, and each operational taxonomic unit was found to be phylogenetically cohesive. Phylogenetic analyses of the sequence data indicated the presence of a broad range of bacteria related to the cyanobacteria, Thermus species, thiobacilli, planctomycetes, thermophilic hydrogen oxidisers, thermotogales, clostridia, actinomycetes, and β and δ subclasses of the proteobacteria. Use of the restriction enzyme analysis protocol also enabled the extent of insert repetition within the library to be monitored, and would thus have eliminated 70% of sequencing expenses. This is the first time a community analysis has been performed on this extreme environment.