Sporotrichosis is one of the most frequent subcutaneous fungal infections to human and animals caused by members of the plant-associated, dimorphic genus Sporothrix. Three of the four medically important Sporothrix species found in Brazil have been considered asexual, as no sexual stage has ever been reported in S. schenckii, S. brasiliensis or S. 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 and to determine the mating type ratio in these pathogens populations. Our analysis revealed the presence of a MAT1-1 locus in S. schenckii while a MAT1-2 locus was found in the S. brasiliensis presenting genomic synteny to other Sordariomycetes. Furthermore, the components of the MAPK-pheromone pathway, pheromone processing enzymes as well as meiotic regulators have also been identified in the two pathogens, suggesting the potential for sexual reproduction. The MAT1-1:MAT1-2 ratio was not significantly different from 1:1 all three Sporothrix species, but the population of S. brasiliensis in the outbreaks was originated from a single mating type. We also explored the population genetic structure of these pathogen populations using sequence data of two loci to improve our knowledge on the pattern of geographic distribution, genetic variation and virulence phenotypes. Population genetics data showed significant population differentiation and clonality with low level of haplotype diversity in S. brasiliensis from different regions of sporotrichosis outbreaks in Brazil. In contrast, S. schenckii 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.