Three real-time PCR–based assays for the specific diagnosis of flavescence dorée (FD), bois noir (BN) and apple proliferation (AP) phytoplasmas and a universal one for the detection of phytoplasmas belonging to groups 16Sr-V, 16Sr-X and 16Sr-XII have been developed. Ribosomal-based primers CYS2Fw/Rv and TaqMan probe CYS2 were used for universal diagnosis in real-time PCR. For group-specific detection of FD phytoplasma, ribosomal-based primers fAY/rEY, specific for 16Sr-V phytoplasmas, were chosen. For diagnosis of BN and AP phytoplasmas, specific primers were designed on non-ribosomal and nitroreductase DNA sequences, respectively. SYBR® Green I detection coupled with melting curve analysis was used in each group-specific protocol. Field-collected grapevines infected with FD and BN phytoplasmas and apple trees infected with AP phytoplasma, together with Scaphoideus titanus, Hyalesthes obsoletus and Cacopsylla melanoneura adults, captured in the same vineyards and orchards, were used as templates in real-time PCR assays. The diagnostic efficiency of each group-specific protocol was compared with well-established detection procedures, based on conventional nested PCR. Universal amplification was obtained in real-time PCR from DNAs of European aster yellows (16Sr-I), elm yellows (16Sr-V), stolbur (16Sr-XII) and AP phytoplasma reference isolates maintained in periwinkles. The same assay detected phytoplasma DNA in all test plants and test insect vectors infected with FD, BN and AP phytoplasmas. Our group-specific assays detected FD, BN, and AP phytoplasmas with high efficiencies, similar to those obtained with nested PCR and did not amplify phytoplasma DNA of other taxonomic groups. Melting curve analysis was necessary for the correct identification of the specific amplicons generated in the presence of very low target concentrations. Our work shows that real-time PCR methods can sensitively and rapidly detect phytoplasmas at the universal or group-specific level. This should be useful in developing defence strategies and for quantitative studies of phytoplasma–plant–vector interactions.