Cases of meningoencephalitis have increased in the zoonotic hyperendemia of sporotrichosis in the state of Rio de Janeiro, Brazil. The gold standard for the diagnosis of sporotrichosis is the isolation of Sporothrix spp. from clinical specimens, not always possible from the cerebrospinal fluid (CSF) of these patients, since fungus recovery in this specimen is difficult in most cases. This limitation led us to pursue a new approach on central nervous system (CNS) sporotrichosis diagnosis based on existing molecular methodologies for the detection of Sporothrix spp. in skin samples. Kano and colleagues (2003) designed species-specific primers for polymerase chain reaction (PCR) based on Sporothrix schenckii sensu lato chitin synthase 1 (CHS1) gene sequence and applied it in skin biopsy paraffin block. Hu and colleagues (2003) used a nested PCR in human clinical samples and samples from infected mice, with the 18S rRNA gene sequence as target. The assay was successfully used to detect S. schenckii DNA from strains from different areas of the world. However, Mendoza and colleagues compared the previously described nested PCR with conventional diagnostic methods, and the molecular methodology presented lower efficacy. Liu and colleagues, using the primer pair S2-R2 targeting the CHS1 gene in the PCR of biopsy tissue, verified positive results in 25 out of 30 cases (83.3%). The nested PCR targeting the partial sequence of the 18S rRNA gene was the best choice in terms of sensitivity due to a low fungal burden in CSF.