Technical Abstract: Panama disease of banana, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense, is a serious constraint both to the commercial production of banana and cultivation for subsistence agriculture. Previous work has suggested that F. oxysporum f. sp. cubense consists of several clonal lineages that may be genetically distant. In this study we test if lineages of the Panama disease pathogen have an exclusive common origin by comparing DNA sequences of nuclear and mitochondrial genes. DNA sequences were obtained for translation elongation factor 1 alpha (EF-1 alpha) and the gene for mitochondrial small subunit (mtSSU) ribosomal RNA for F. oxysporum strains from banana, pathogenic strains from other hosts and nonpathogenic isolates of the fungus. Cladograms for the two genes were highly concordant and a partition-homogeneity test indicated the two datasets could be combined. The combined tree resolved five taxa corresponding to "F. oxysporum f. sp. cubense" with a large dichotomy between the two taxa representing cultures most commonly isolated from bananas with Panama disease. F. oxysporum isolates collected as nonpathogenic or pathogenic to other hosts may have very similar or identical EF-1 alpha and mtSSU genotypes as banana pathogens, yet cause little or no disease on banana. Strains in the two largest taxa from banana have significantly different chromosome numbers. Taken together these results indicate Panama disease is caused by fungi with separate evolutionary origins.