Panama disease of banana, caused by the fungus Fusarium oxysporum f. sp. cubense, is a serious constraint both to the commercial production of banana and cultivation for subsistence agriculture. Previous work has indicated that F. oxysporum f. sp. cubense consists of several clonal lineages that may be genetically distant. In this study we tested whether lineages of the Panama disease pathogen have a monophyletic origin by comparing DNA sequences of nuclear and mitochondrial genes. DNA sequences were obtained for translation elongation factor 1alpha and the mitochondrial small subunit ribosomal RNA genes for F. oxysporum strains from banana, pathogenic strains from other hosts and putatively nonpathogenic isolates of F. oxysporum. Cladograms for the two genes were highly concordant and a partition-homogeneity test indicated the two datasets could be combined. The tree inferred from the combined dataset resolved five lineages corresponding to "F. oxysporum f. sp. cubense" with a large dichotomy between two taxa represented by strains most commonly isolated from bananas with Panama disease. The results also demonstrate that the latter two taxa have significantly different chromosome numbers. F. oxysporum isolates collected as nonpathogenic or pathogenic to other hosts that have very similar or identical elongation factor 1alpha and mitochondrial small subunit genotypes as banana pathogens were shown to cause little or no disease on banana. Taken together, these results indicate Panama disease of banana is caused by fungi with independent evolutionary origins.