Abstract: Objective To evaluate the sensitivity and specificity of primers for Sporothrix schenckii. Methods Genomic DNA was extracted from 50 strains of S.schenckii, including 49 clinical isolates and 1 referrence strain, as well as from 1 strain of Mucor, Aspergillus fumigatus and Candida albicans, respectively, as controls. Four primers were designed according to previous studies, and screened by PCR. Specific primers were defined as those capable of yielding the same DNA fragment from all strains of S.schenckii, but not from any control fungal strains. Then, the genomic DNA was proportionally diluted and subjected to PCR amplification. The minimal concentration of template DNA required for efficient amplification was determined for the sensitivity assay of primers. Results Under the same PCR conditions, three primers, including S2-R2, SSHF31-SSHR97 and ITS3-SSP, had a relatively high specificity. PCR with the primer SS3-SS4 amplified the same DNA fragment from S.schenckii and C.albicans strains. The primer S2-R2 had the highest sensitivity with a detection limit of 5 pg/μL. Conclusion So far, the primer S2-R2 targeting the chitin synthase 1 gene is the most specific and sensitive primer for S.schenckii .