In 2010 and 2011 surveys of Fars provience were observed symptoms doubtful to phytoplasma diseases such as virescence and phyllody, floral proliferation, witches’-broom, yellowing and little leaf in eggplant fields. To determination of etiology this symptoms, seven infected eggplant plants from Fasa, Jahrom, Firouzabad, Darab and Sarvestan were planted in suitable pots, transferred to a greenhouse without any insects and used as sources of disease for further studies. The causal agents were transmitted from diseased to healthy eggplants by grafting and from eggplant to periwinkle by dodder. In inoculated eggplants, were appeared big bud, little leaf, yellowing and witches’-broom and in inoculated periwinkles, were appeared virescence, yellowing and witches’-broom. Total DNA were extracted from fresh midrib tissues of naturally infected eggplant, experimentally inoculated periwinkle plants and a healthy eggplant and periwinkle plant. In direct PCR using primer pairs P1/P7, DNA fragments of approximately 1800 bp were amplified from total nucleic acid samples extracted from symptomatic eggplant and periwinkle plants. Periwinkle reaction to inoculation with Eggplant Big Bud (EBB) different isolates was variable as based on difference in severity and type of symptoms in periwinkle, EBB different isolates, were placed in six groups. Direct PCR products of EBB different isolates were digested with AluI, HaeIII and RsaI enzymes. Comparison of RFLP patterns indicated that EBB different isolates place in two groups: Fasa 1, Fasa 2, Fasa 3, Firouzabad, Darab and Sarvestan isolates in one group and Jahrom isolate in other group. Based on disease symptoms, dodder and graft transmission and positive reaction in PCR, eggplant big bud in Fars province has phytoplasmal etiology. This is the first report of the presence of EBB in Fasa, Firouzabad, Darab and Sarvestan. With molecular testings in this study, were confirmed there are at least two phytoplasmas associated with EBB in Fars province. By using more restriction enzymes, it is likely to see more diversity in EBB agent in Fars province.