Sporotrichosis is the most prevalent subcutaneous mycosis worldwide and increasing number of cases is being reported in the last decade. It is caused by thermodimorphic fungi of the Sporothrix schenckii complex and affects immunosuppressed and immunocompetent individuals. S. brasiliensis is the main causing agent of this mycosis in Brazil, where sporotrichosis is hyperendemic. This organism presents an increased virulence and a highly aggressive phenotype, with an increasing antifungal resistance. As a matter of fact, the earlier and proper diagnosis of the causing agent, in case of sporotrichosis infection, is important for its efficient treatment. Therefore, the unravelling of the virulence mechanisms of S. brasiliensis is of major importance in order to improve the diagnosis and treatment of sporotrichosis. During this work, we aimed to develop a molecular toolkit for the unravel of S. brasiliensis virulence traits. Our results from flow cytometry (FCM) for ploidy analysis of S. brasiliensis, S. schenckii, S. globosa, S. pallida and S. mexicana point to a diploid DNA content for these Sporothrix spp. Moreover, using bioinformatic tools, we performed a ploidy estimation using next-generation sequencing (NGS) data, which is in accordance with our FCM results. In the context of this work, we also developed an efficient protocol for transformation of S. brasiliensis using Agrobacterium tumefaciens. The application of this protocol resulted in 7851±3572 clones/co-cultivation, using a 3:1 ratio (A. tumefaciens:S. brasiliensis) at 27ºC for 72 h. The clones were mitotically stable, allowing the future utilization of this methodology to produce libraries of insertional mutants for identification of virulence factors. Additionally, we established an in vitro model of S. brasiliensis infection using human monocyte derived macrophages (MDMs), aiming, in the future, to analyse the transcriptome profile by dual RNA-seq of both fungi and infected macrophages. In this sense, infection conditions were optimized for more than 99% of cells internalization/adherence. This was achieved for a multiplicity of infection (MOI) of 1:3 (macrophages:fungi) for 2 h, and at least 80% of fungal cells and macrophages remained viable post-infection. These conditions were also tested for S. schenckii and similar results were obtained, allowing the comparison of the transcriptomic profile between these two organisms. This is expected to contribute for the future study of S. brasiliensis, particularly for the disclosure of its virulence traits, as well as the study of the immune response of macrophages to these fungi. This response was preliminary explored in the context of this work and MDMs presented similar fungicidal activity against S. schenckii and S. brasiliensis, and S. schenckii exhibited a more pro-inflammatory profile, with increased production of interleukin (IL)- 1Ⱦ and tumor necrosis factor (TNF)-Ƚ.