Sporothrix (Ophiostomatales) comprises species that are pathogenic to humans and other mammals as well as environmental fungi. Developments in molecular phylogeny have changed our perceptions about the epidemiology, host-association, and virulence of Sporothrix. The classical agent of sporotrichosis, Sporothrix schenckii, now comprises several species nested in a clinical clade with S. brasiliensis, S. globosa, and S. luriei. To gain a more precise view of outbreaks dynamics, structure, and origin of genetic variation within and among populations of Sporothrix, we applied three sets of discriminatory AFLP markers (#3 EcoRI-GA/MseI-TT, #5 EcoRI-GA/MseI-AG, and #6 EcoRI-TA/MseI-AA) and mating-type analysis to a large collection of human, animal and environmental isolates spanning the major endemic areas. A total of 451 polymorphic loci were amplified in vitro from 188 samples, and revealed high polymorphism information content (PIC = 0.1765-0.2253), marker index (MI = 0.0001-0.0002), effective multiplex ratio (E = 15.1720-23.5591), resolving power (Rp = 26.1075-40.2795), discriminating power (D = 0.9766-0.9879), expected heterozygosity (H = 0.1957-0.2588), and mean heterozygosity (Havp = 0.000007-0.000009), demonstrating the effectiveness of AFLP markers to speciate Sporothrix. Analysis using the program structure indicated three genetic clusters matching S. brasiliensis (population 1), S. schenckii (population 2), and S. globosa (population 3), with the presence of patterns of admixture amongst all populations. AMOVA revealed highly structured clusters (PhiPT = 0.458-0.484, P < 0.0001), with roughly equivalent genetic variability within (46-48 %) and between (52-54 %) populations. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for S. schenckii (χ2 = 2.522; P = 0.1122), supporting random mating. In contrast, skewed distributions were found for S. globosa (χ2 = 9.529; P = 0.0020) with a predominance of MAT1-1 isolates, and regional differences were highlighted for S. brasiliensis with the overwhelming occurrence of MAT1-2 in Rio de Janeiro (χ2 = 14.222; P = 0.0002) and Pernambuco (χ2 = 7.364; P = 0.0067), in comparison to a higher prevalence of MAT1-1 in the Rio Grande do Sul (χ2 = 7.364; P = 0.0067). Epidemiological trends reveal the geographic expansion of cat-transmitted sporotrichosis due to S. brasiliensis via founder effect. These data support Rio de Janeiro as the centre of origin that has led to the spread of this disease to other regions in Brazil. Our ability to reconstruct the source, spread, and evolution of the ongoing outbreaks from molecular data provides high-quality information for decision-making aimed at mitigating the progression of the disease. Other uses include surveillance, rapid diagnosis, case connectivity, and guiding access to appropriate antifungal treatment.