Sugarcane white leaf disease (SCWL) is one of the most destructive diseases of sugarcane in Thailand. An efficient and reliable method to detect the pathogen is urgently needed for the development of disease control methods. DNA of the causal agent, a phytoplasma, was isolated, random DNA fragments cloned and used as DNA probes to detect the pathogen. DNA of the SCWL phytoplasma was isolated from host DNA by repeated bisbenzimide-CsCl equilibrium density gradient centrifugation. The DNA fragments cut by restriction enzyme HindIII were cloned in a plasmid-Escherichia coli system. Southern hybridization analysis revealed that the inserts of some recombinant plasmids consisted of fragments of chromosomal DNA of the phytoplasma, whereas the inserts of the other recombinant plasmids consisted of fragments of extrachromosomal DNA of the phytoplasma. Cloned DNA probes were successfully applied in dot hybridization to detect the phytoplasma in sugarcane plants and in the insect vector Matsumuratettix hiroglyphicus. Dot hybridization using the DNA probes indicated that SCWL and phytoplasma of gramineous plants including rice yellow dwarf phytoplasma shared a greater nucleotide sequence homology with one another than with other phytoplasma.