Tomato big bud was detected for the first time in tomato plants (Lycopersicon esculentum Mill.) in the eastern region (Al-Mafraq) of Jordan. Infected plants showed proliferation of lateral shoots, hypertrophic calyxes and greening of flower petals. The presence of phytoplasmas in diseased tomato plants was demonstrated using polymerase chain reaction (PCR) assays. The amplified DNAs yielded products of 1.8 kb (primer pair P1/P7) and 1.2 kb (primer pair R16F2/R2) by direct and nested-PCR, respectively. DNA from tomato isolates T1 and T2 could not be amplified in the nested-PCR assays when the aster yellow-specific primer pair R16(1)F1/R1 was used, suggesting that the phytoplasma in these isolates is not genetically related to the 16SrI (aster yellows) group. After restriction fragment length polymorphism (RFLP) analyses, using four endonuclease enzymes (HhaI, RsaI, AluI and Bsp143I) similar patterns were formed among the digested 1.2 kb PCR products of two tomato isolates suggesting that both isolates belonged to the same phytoplasma. Compared with the RFLP profile of the reference strains, no difference in the digestion pattern was found between the tomato isolates and that of the catharanthus phyllody agent from Sudan, indicating that the phytoplasma belongs to 16SrDNA VI (clover proliferation) group.