End-point PCR, followed by restriction fragment length polymorphism (RFLP) or sequencing and sequence analysis of the 16S rRNA gene, is a cornerstone method for the universal detection and identification of phytoplasmas, including previously undescribed strains. This protocol describes the use of end-point PCR in direct, nested, or semi-nested systems, coupled with RFLP analysis using 17 restriction enzymes, to classify phytoplasmas into distinct 16Sr ribosomal groups and subgroups while addressing challenges such as inter-operon heterogeneity and mixed infections. Alternatively, sequencing of the PCR-amplified 16S rRNA gene, followed by sequence analysis (e.g., virtual RFLP or sequence homology), enables classification into either 16Sr groups/subgroups or ‘Candidatus Phytoplasma' species. The method’s sensitivity, cost-effectiveness, and compatibility with established classification frameworks make it invaluable for epidemiological studies, quarantine measures, and the delineation of ‘Candidatus Phytoplasma' species. By providing a clear framework for the precise diagnosis of phytoplasma-associated diseases in diverse plant and insect hosts, this protocol supports rapid responses to outbreaks and helps mitigate the economic impact of phytoplasmas.