A molecular karyotype of the corn smut-inducing fungus Ustilago maydis was prepared using orthogonalfield-alternation gel electrophoresis (OFAGE). At least 20 chromosome-sized DNAs ranging from approximately 300 kb to the maximum limit of resolution of this system were identified in haploid cells of a widely used strain. Although general features of the banding pattern of chromosome-sized DNAs were conserved between strains, no two strains had identical karyotypes, indicating that considerable chromosome length polymorphism exists in this species. This polymorphism was seen in both laboratory strains as well as more recent isolates from nature. Length variation in apparently identical chromosomes was usually small, but was occasionally significant. In one strain Southern DNA hybridization analysis suggested the occurrence of a stable large-scale, inter-chromosomal exchange which had given rise to two novel chromosomes.