ABSTRACT To better understand the nature of recent outbreaks of leaf scald disease of sugarcane in a number of sugarcane production regions of the world including Florida, Guadeloupe, Louisiana, Mauritius, Taiwan, and Texas, a study of the worldwide genetic variation of the pathogen was undertaken. A total of 218 strains from 31 geographic locations were examined. Genomic DNA of each strain was digested with the rare cutting restriction enzyme SpeI, and the fragments were separated by pulsed-field gel electrophoresis (PFGE). A total of 102 bands were identified, and 54 different DNA banding patterns (haplotypes) were observed. Eight groups of banding patterns, designated PFGE groups A through H, were consistently detected by visual, principal component, and cluster analyses. Five groups were comprised of multiple haplotypes representing numerous strains, and three were comprised of single haplotypes representing one strain each. The leaf scald outbreaks in Florida, Louisiana, Texas, and possibly Guadeloupe and Taiwan could be attributed to the introduction of strains belonging to PFGE group B. When infection by two strains each of the newly introduced strains (PFGE group B) and those previously present in Florida (PFGE group A) was analyzed in 22 sugarcane cultivars by reisolation 24 weeks after inoculation, a significantly greater mean frequency was detected for PFGE group B strains and no cultivar by PFGE group interaction was observed. Inadvertent dispersal of the pathogen among plants, possibly by means of aerosols or splashing water, was detected in a subsequent experiment. Strains of PFGE group B were recovered from the internal tissues of some plants inoculated with PFGE group A strains and were also found to be epiphytic colonizers of nonsymptomatic, noninoculated plants adjacent to the inoculated plants; whereas strains of PFGE group A were recovered only from plants that had been inoculated with them. Thus, the possibility became more apparent that strain variation might be associated, at least in part, with factors governing plant-to-plant spread of the pathogen in nature.