We studied 23 clinical and environmental strains of Sporothrix schenckii sensu lato collected from 1984 to 2017 in Argentina. The molecular identification (partial sequencing of a fragment of the calmodulin gene) of the strains was performed. For the yeast and mycelial phases, the in vitro susceptibility testing by a microdilution reference method was determined against eight antifungal drugs. Strains studied were identified as S. schenckii sensu stricto 13 (56.5%), S. brasiliensis 8 (34.7%) and S. globosa 2 (8.7%). The most active antifungal drugs tested for the yeast and mycelial phases expressed as geometric mean (GM) value of the minimal inhibitory concentration (MIC) (μg mL ) were terbinafine (0.07 and 0.24), posaconazole (0.13 and 0.58), itraconazole (0.38 and 1.10) and ketoconazole (0.22 and 0.89), while fluconazole (110.10 and 131.92) and flucytosine (2.96 and 79.03) were the less active. For voriconazole and amphotericin B the GM-MIC values were acceptably low for the yeast phase (0.39 and 0.72 μg mL ), while the mycelial phase showed values ≥2-fold higher (8.76 and 1.88 μg mL ), P < .05. Here, we described S. schenckii sensu stricto, S. brasiliensis and S. globosa, these species were isolated from humans, animals and soil and are circulating in Argentina since at least 1984.