This study describes a phytoplasmal disease occurring in Petunia leaves grown in the glasshouse of the National Horticultural Research Institute, Suwon, Korea. Abnormal growth like flattened stem with flower malformation or phyllody was observed from the plant. The DNA extracted from the diseased leaves was amplified using a universal primer pair of P1/P6 derived from the conserved 16S rRNA gene of Mollicutes giving the expected polymerase chain reaction (PCR) product of 1.5 kb. In the nested PCR assays, the expected DNA fragment of 1.1 kb was amplified with the specific primer pair R16F1/R16R1 that was designed on the basis of aster yellows (AY) phytoplasma 16S rDNA sequences. The 1.1 kb PCR products were cloned and nucleotide sequences were determined, and the sequences of the cloned 16S rRNA gene were deposited in the GenBank database under the accession no. of EU267779. Analysis of the homology percent of the 16S rDNA of PFS-K showed the closest relationship with Hydrangea phyllody phytoplasma (AY265215), Brassica napus phytoplasma (EU123466) and AY phytoplasma CHRY (AY180956). Phytoplasma isolated from the diseased Petunia was designated as Petunia flat stem phytoplasma Korean isolate (PFS-K) in this study. Flattened stem occurring in Petunia was confirmed as infection of AY group of phytoplasma by determination of 16S rRNA gene sequences of phytoplasma and microscopic observation of phytoplasma bodies. This is the first report on the phytoplasmal disease in Petunia in Korea.