has emerged as the most virulent species in the complex, accounting for sporotrichosis. Albeit the new insights into the understanding of host-pathogen interactions and comparative genomics of this fungi, the lack of genetic tools has hindered significant advances in this field of research. Here, we established an Agrobacterium tumefaciensmediated transformation (ATMT) system to transform different strains of . We report parameters that account for a transformation efficiency of 3,179 ± 1,171 transformants/co-cultivation, which include the use of A. tumefaciens AGL-1 in a 2:1 ratio (bacteria:fungi) during 72 h at 26°C. Our data show that a single-copy transgene is transferred to that is mitotically stable in 99% of cells after 10 generations without selective pressure. In addition, we created a plasmid toolkit that allows the establishment of fusion proteins of any gene of interest with sGFP or mCherry under the control of the GAPDH or H2A endogenous promoters. These modules allow different levels of expression of the desired fusion. Moreover, we successfully targeted these fluorescent proteins to the nucleus and used fluorescence-tagged strains to assess phagocytosis. Overall, our data show that the ATMT system is an easy-to-use and efficient genetic toolbox for studies on recombinant expression and gene function in . Sporotrichosis is the most prevalent subcutaneous mycosis worldwide and has recently become a public health concern. Although immunocompetent hosts are also prone to sporotrichosis, immunodeficient hosts often develop a more severe and disseminated form of disease. To date, the Rio de Janeiro state in Brazil is the most significant feline zoonotic transmission epicenter in the world, with more than 4,000 human and feline diagnosed cases. Cats play an essential role in the infection due to their high susceptibility and transmissibility to other felines and humans. is the most virulent etiological agent of sporotrichosis, causing the most severe clinical manifestations. Despite the increasing incidence of sporotrichosis, the identification of virulence traits important for disease establishment, development, and severity has been lacking. In this work, we established an efficient genetic toolbox to manipulate that will guide future studies to define new virulence mechanisms and a better understanding of host-pathogen interactions from a molecular perspective.