This study examined whether genes that are less conserved than the 16S rRNA gene can distinguish Candidatus Phytoplasma australiense strains that are identical based on their 16S rRNA genes, with a view to providing insight into their origins and distribution, and any patterns of association with particular plant hosts. Sequence analysis of the tuf gene and rp operon showed that Ca. P. australiense strains could be differentiated into four subgroups, named 16SrXII-B (tuf-Australia I; rp-A), 16SrXII-B (tuf-New Zealand I; rp-B), 16SrXII-B (tuf-New Zealand II) and 16SrXII-B (rp-C). Strawberry lethal yellows 1, strawberry green petal, Australian grapevine yellows, pumpkin yellow leaf curl and cottonbush witches' broom phytoplasmas were designated members of the 16SrXII-B (tuf-Australia I; rp-A) subgroup. The strawberry lethal yellows 2 and cottonbush reduced yellow leaves phytoplasmas were assigned to the 16SrXII (tuf-New Zealand II; rp-B) subgroup. No relationship was observed between these phytoplasma subgroups and collection date, location or host plant. However, the study revealed evolutionary divergence in the 16SrXII group.