Genomic clones hybridizing to anonymous, single-copy sequences were used to probe chromosome-sized DNAs of Fusarium oxysporum f. sp. cubense separated by pulsed-field gel electrophoresis. As expected, most clones hybridized to single chromosome bands. However, two of eight "single-copy" clones hybridized to two chromosomes in some, but not all, of 14 isolates examined. This observation suggests a degree of genetic duplication in the fungus and is consistent with recent electrophoretic karyotype analysis indicating that intraspecific differences in genome size and chromosome number may be explained, at least in part, by persistent genetic duplication.