An environmental survey of tabanid host spiroplasma carriage was undertaken at 10 collection sites in Australia during February 1999. A total of 164 tabanid flies, representing 27 species, were collected and sustainable spiroplasma isolations were made from 48 of the flies. The morphology of the cultured spiroplasmas, as observed in M1D medium under dark-field microscopy, was typical of either (i) Apis group spiroplasmas (relatively thick cells (~150 nm) with six or more turns) or (ii) chrysopicola–syrphidicola–TAAS-1 clade spiroplasmas (narrower, often much shorter cells) serologically related to Spiroplasma serogroup VIII. Repetitive serological analyses, involving successive rounds of dilution cloning and serological reevaluation, identified one serotype referable to the Spiroplasma serogroup VIII strain complex and five putative members of the Apis clade. Apis clade placement for these five groups was verified using 16S rRNA phylogenetic analyses. Among the Apis clade members, one serotype representing 11 isolates was identified as a geographic variant of Spiroplasma turonicum. Spiroplasma turonicum (Tab4C) was originally isolated from a tabanid Haematopoda sp. in France. The other 34 isolates represented four new serogroups (= putative species). The following strains are proposed as representatives of the new serogroups: strain GSU5478 (group XXXIX), strain GSU5490 (group XL), strain GSU5508 (group XLI), and strain GSU5603 (group XLII). In summary, six serogroups were observed from isolations originating from seven distinct sample sites in Australia. Surprisingly, the serotype with the greatest geographical range (five sites from 16°48.9′S to 35°40.0′S) and the greatest host diversity (nine species over three genera) was the geographic variant of S. turonicum, which had only been reported previously in France.