Phytoplasmas are cell wall-less bacteria that cause numerous diseases in several hundred plant species. During adaptation to transkingdom parasitism in diverse plant and insect hosts, phytoplasma evolution has given rise to widely divergent lineages. Since phytoplasmas cannot be cultured in a cell-free medium, measurable phenotypic characters suitable for conventional microbial classification are mostly inaccessible. Currently, phytoplasma differentiation and classification are mainly dependent on restriction fragment length polymorphism (RFLP) analysis of 16S rRNA gene sequences. Extending our recent efforts in the exploitation of computer-simulated 16S rRNA gene RFLP analysis and virtual gel plotting for rapid classification of phytoplasmas, we have developed a Perl program for automated RFLP pattern comparison and similarity coefficient calculation. This program streamlines virtual RFLP pattern analysis and has led to the establishment of a criterion for phytoplasma 16Sr subgroup classification and to the delineation of new and distinct subgroup lineages in the clover proliferation phytoplasma group (16SrVI).