Molecular diversity among clinical isolates of Madurella mycetomatis, the prime fungal agent of human mycetoma in Sudan, could possibly explain the diverse clinical presentations of this severely debilitating infectious disease. In addition, culture-independent DNA-mediated typing tests need to be developed for this organism, since M. mycetomatis DNA, but not the organism itself, can be identified in soil, the material from which infections are thought to originate. A collection of 38 different clinical M. mycetomatis isolates was characterized by large-scale random amplification of polymorphic DNA using 20 different primer species. These analyses, involving at least 2,600 annealing sites, showed a complete lack of DNA fingerprint variation among the various isolates. From the resulting homogeneous DNA fingerprints, seven fragments were cloned and sequenced, and novel, species-specific PCR restriction fragment length polymorphism (RFLP) tests were designed. The seven PCR RFLP tests were successfully performed on the 38 different M. mycetomatis strains. However, again all M. mycetomatis DNA patterns obtained appeared to be identical, whereas patterns produced using DNAs from other fungal species were clearly discriminatory. These results suggest that there is little genetic variation among clinically relevant M. mycetomatis strains from Sudan. The data tentatively imply that different manifestations of mycetoma are due to differences in host susceptibility rather than differential virulence of the causative agent.