Two techniques were developed for the analysis of non-cultivable mollicutes in insects. The first was aimed at detecting organisms belonging to undiscovered groups within the phytoplasma clade. After prescreening by polymerase chain reaction with phytoplasma-specific primers, nucleic acids from 54 positive samples were amplified using phytoplasma-specific fluorescein-labelled primers flanking the 16S-23S rDNA spacer region, which is variable in length among the phytoplasmas. The sizes of all the detected products were only those expected for already-described phytoplasma subclades. It was also shown that a single leafhopper might carry different phytoplasmas, at similar or very different relative concentrations. The second technique, based on the heteroduplex mobility assay, was designed for the detection of organisms phylogenetically similar to phytoplasmas but not recognized by the specific primer pair. As a result, signals generated by ribosomal DNA of organisms which appear to be closely related but not identical to phytoplasmas were detected.