Detection and classification of phytoplasmas mainly rely on amplification of the 16S rRNA gene followed by RFLP analysis and/or sequencing, because these organisms lack complete phenotypic characterization. Other conserved genomic loci have been exploited as additional molecular markers for phytoplasma differentiation. Two loci, SSU12p and LSU36p, selected by whole-genome comparison of 12 phytoplasma strains, were used for primer design, and were successfully tested on DNA samples from plants infected by phytoplasmas belonging to ten 16S ribosomal groups. The phylogenetic trees inferred from SSU12p and LSU36p loci were highly congruent to the trees derived from 16S rRNA and tuf genes of the same phytoplasma strains. Virtual RFLP analysis of the amplified SSU12p gene showed distinct patterns for most of the phytoplasma ribosomal subgroups tested. These results show that SSU12p and LSU36p genes are reliable additional markers for phytoplasma detection and differentiation.