Sporothrix schenckii and related species are the agents of human and animal sporotrichosis. Routine diagnoses using classical mycological approaches are unspecific due to overlapping phenotypes. As the frequency and prevalence of sporotrichosis increases worldwide, developing specific, sensitive and cost-effective diagnostic tools is essential to understand the distribution patterns, map-affected areas and promote specific public health strategies to mitigate future outbreaks. Polymorphisms among the β-tubulin gene were exploited to speciate S. brasiliensis, S. schenckii and S. globosa in a one-tube multiplex probe-based qPCR assay. A panel of 84 Sporothrix revealed 100% specificity (AUC = 1.000, 95% CI = 0.971-1.000, p < .0001) without cross-reacting with other medically relevant fungi, human, feline or murine DNA. Speciation via multiplex qPCR matched phylogenetic identification (Kappa = 1.0; 95% CI = 1.0-1.0; very good agreement), supporting its use as a reliable alternative to DNA sequencing. Remarkably, the lower limit of detection was 3 copies of the target for all species. As a proof of concept, we used swabs of wound exudate of 70 cats suspected of sporotrichosis to reveal an overwhelming occurrence of S. brasiliensis in 69 specimens (sensitivity = 98.57%; 95%CI: 92.3-100.0 and specificity = 100%; 95% CI = 78.2-100). In comparison to culture, qPCR showed a larger area under the curve (AUC = 0.993±0.007; 95% CI = 0.944-1.000; p < .0001; Youden's index = 0.9857), supporting that qPCR is an essential tool for accurately detect Sporothrix DNA directly from clinical samples, thus accelerating the diagnosis of sporotrichosis. Moreover, our multiplex qPCR system has the potential to increase diagnostic capacity in Sporothrix-affected areas, helping the local animal health agent or veterinarian to quickly identify and isolate new cases, which will likely benefit thousands of patients infected every year worldwide.