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ABSTRACTSporotrichosis is one of the most frequent subcutaneous fungal infections in humans and animals caused by members of the plant-associated, dimorphic genus Sporothrix. Three of the four medically important Sporothrixspecies found in Brazil have been considered asexual as no sexual stage has ever been reported in Sporothrix schenckii, Sporothrix brasiliensis, or Sporothrix globosa. We have identified the mating type (MAT) loci in the S. schenckii(strain 1099-18/ATCC MYA-4821) and S. brasiliensis(strain 5110/ATCC MYA-4823) genomes by using comparative genomic approaches to determine the mating type ratio in these pathogen populations. Our analysis revealed the presence of a MAT1-1locus in S. schenckiiwhile a MAT1-2locus was found in S. brasiliensisrepresenting genomic synteny to other Sordariomycetes. Furthermore, the components of the mitogen-activated protein kinase (MAPK)-pheromone pathway, pheromone processing enzymes, and meiotic regulators have also been identified in the two pathogens, suggesting the potential for sexual reproduction. The ratio of MAT1-1to MAT1-2was not significantly different from 1:1 for all three Sporothrixspecies, but the population of S. brasiliensisin the outbreaks originated from a single mating type. We also explored the population genetic structure of these pathogens using sequence data of two loci to improve our knowledge of the pattern of geographic distribution, genetic variation, and virulence phenotypes. Population genetics data showed significant population differentiation and clonality with a low level of haplotype diversity in S. brasiliensisisolates from different regions of sporotrichosis outbreaks in Brazil. In contrast, S. schenckiiisolates demonstrated a high degree of genetic variability without significant geographic differentiation, indicating the presence of recombination. This study demonstrated that two species causing the same disease have contrasting reproductive strategies and genetic variability patterns.