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Madurella mycetomatis is one of the main causative agents of mycetoma, a debilitating neglected tropical disease. Improved understanding of the genomic diversity of the fungal and bacterial causes of mycetoma is essential to advances in diagnosis and treatment. Here, we describe a high-quality genome assembly of M. mycetomatis and results of the whole genome sequence analysis of 26 isolates from Sudan. We demonstrate evidence of at least seven genetically diverse lineages and extreme clonality among isolates within these lineages. We also performed shotgun metagenomic analysis of DNA extracted from mycetoma grains and showed that M. mycetomatis reads were detected in all sequenced samples with the average of 11,317 reads (s.d. +/- 21,269) per sample. In addition, 10 (12%) of the 81 tested grain samples contained bacterial reads including Streptococcus sp., Staphylococcus sp. and others. Author summary: Mycetoma is a debilitating disease recognized as a neglected tropical disease by the World Health Organization. The etiology of mycetoma is poorly understood; approximately 60% of cases are caused by fungi and the rest are bacterial, although this varies by region. The pathogenic fungus, Madurella mycetomatis, is most frequently identified from mycetoma cases. Here, we present a high-quality genome assembly of M. mycetomatis and results of the whole genome sequence analysis of 26 isolates from Sudan. We demonstrate evidence of at least seven genetically diverse lineages and extreme clonality among isolates within these lineages. Shotgun metagenomic analysis of DNA from mycetoma grains confirmed that M. mycetomatis was the predominant causative agent of eumycetoma Sudan; however, 10% of grains also contained bacterial reads. Thorough understanding of the genetic structure and diversity of fungi causing mycetoma is essential for the development of new diagnostic methods and identifying potential drug targets. [ABSTRACT FROM AUTHOR]