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.