Recently, Russian olive trees showing witches’-broom and little leaf symptoms have been widely observed in northwestern and central Iran. Polymerase chain reaction (PCR) and nested PCR assays using phytoplasma universal primer pairs confirmed phytoplasma symptomatic infection of trees. Sequence analyses showed that ‘Candidatus Phytoplasma asteris’ was the causal agent of the disease in these regions. However, RFLP results using restriction enzymes HpaII, EcoRI, HinfI and AluI indicated that the collected isolates in these regions are genetically different. In addition, leafhopper Macropsis infuscata was recognized as a possible insect vector of the disease for the first time.