Sugarcane white leaf disease (SCWL) is one of the most destructive sugarcane diseases in Thailand. The disease is caused by wall-less microorganisms resembling mycoplasmas, called phytoplasmas. As it has not been possible to culture phytoplasmas in vitro, there has been a lack of information about the phytoplasmas, and it has been difficult to detect the pathogens. DNA of SCWL phytoplasmas was isolated from diseased sugarcane plants and the fragments were cloned in Escherichia coli. By using the cloned DNA fragments as probes, we could detect SCWL phytoplasmas from host plants and insect vectors by a hybridization assay. Rapid detection using PCR also enabled us to detect the ribosomal DNA (rDNA) fragments of the phytoplasmas. Hybridization assay and sequence analysis of rDNA of the phytoplasmas revealed that the genome of the SCWL phytoplasmas and that of the rice yellow dwarf (RYD) phytoplasmas are close to each other. The rDNA analysis showed that the SCWL phytoplasmas are also related to the sugarcane grassy shoot phytoplasmas and other phytoplasmas associated with white leaf diseases in gramineous weeds in Northeast Thailand phylogenetically, but that they are not identical. Sequence analysis of the cloned fragments of the SCWL phytoplasmas revealed that the phytoplasmas have circular 2.6-kb extrachromosomal DNAs. Hybridization assay indicated that the extrachromosomal DNAs of SCWL phytoplasmas and those of the RYD phytoplasmas and phytoplasmas associated with white leaf symptoms in gramineous weeds shared a considerable nucleotide sequence homology, whereas little homology with those of sesame phyllody phytoplasmas and aster yellows-type phytoplasmas. RFLP analysis revealed that the extrachromosomal DNAs showed a polymorphism among isolates collected within an individual field. It is assumed that the DNA-based detection methods and molecular studies on the phytoplasmas may contribute to the development of methods of control of the SCWL disease.