We studied 40 strains of the species complex formerly classified as the single species Sporothrix schenckii to identify new species within this complex and evaluate their antifungal susceptibility profiles. Based on phenotypic tests (ability to grow at 37°C, colony diameters, and pigmentation of the colonies, as well as assimilation of sucrose and raffinose) and molecular assays (amplification of a fragment of the calmodulin gene), here we report the identification of S. albicans, S. brasiliensis, S. luriei, and S. schenckii; two isolates of these species were detected as itraconazole-resistant strains.Sporotrichosis is a subcutaneous mycosis affecting humans and animals caused by Sporothrix schenckii. It has a worldwide distribution, especially in tropical and subtropical areas of Latin America, where areas of endemicity have been recognized (1,3,4,12). Recently, Marimon et al. (9,11). proposed that Sporothrix schenckii is a complex encompassing six cryptic species that had been previously identified by others (4). In this context, variation in the antifungal susceptibility profiles among these new species was hypothesized. The aim of this study was to explore a collection of 40 isolates formerly classified as Sporothrix schenckii in order to identify new species and evaluate their susceptibility to antifungal agents.The isolates were from cases of human (n ϭ 31) and animal (n ϭ 9) sporotrichosis diagnosed in the hinterlands of Rio Grande do Sul (Brazil) and were maintained in the Department of Microbiology of the Universidade Federal de Santa Maria (UFSM), Santa Maria, Brazil. Among the human-derived strains, 18 (58.06%) were from fixed cutaneous sporotrichosis and 13 (41.9%) were from the lymphocutaneous form of the mycosis. Of the strains isolated from animals (n ϭ 9), eight were from cats and one (S. luriei) was isolated from a dog with sporotrichosis. As proposed by Marimon et al. (9,11), the phenotypic tests included the ability/inability to grow at 37°C on potato dextrose agar (PDA; HiMedia, Mumbay, India), different colony diameters (mm) after 21 days of incubation at 30°C on PDA, the pigmentation of the colonies on cornmeal agar (CMA), and the assimilation of sucrose and raffinose. The susceptibility tests were conducted according the procedures proposed for the M38-A2 technique (2). For molecular identification, a fragment of the calmodulin (CAL) gene was amplified from genomic DNA using the degenerate primers CL1[5Ј-GA(GA)T(AT)CAAAGGAGGCCTTCTC-3Ј] and CL2A (5Ј-TTTTTGCATCATGACTTGGAC-3Ј) (9). DNA sequencing was performed on the purified amplicons using a MegaBace 500 automatic sequencer. The sequences were aligned against sequences available in GenBank with ClustalX (version 1.8) followed by manual adjustments with a text editor. The phylogenetic analysis was performed with MEGA (Molecular Evolutionary Genetic Analysis) software version 4.0 (17).Based on recently proposed procedures (9, 11), we phenotypically identified four species: Sporothrix schenckii (n ϭ 37), Sporothrix brasiliensis (n ϭ 1), Sporo...