In this research, the genomic DNA of mating type isolate of Sporisorium scitamineum was used as the template, five impact factors, namely, template DNA, Mg2+, dNTPs, primer and rTaq DNA polymerase, which were the components of PCR reaction system, were optimized respectively by single factor test. Based on the results, L16(45) orthogonal design was applied for establishing the best SCoT-PCR system for Sporisorium scitamineum, of which the total 25 μL reaction system contained 12.50 ng template DNA, 0.17 mmol/L dNTPs, 0.46 μmol/L primer, 1.7 mmol/L, 0.85 U rTaq DNA polymerase and 1×Buffer (Mg2+ free). Furthermore, 10 polymorphic primers were screened out of 30 tested primers based on the above optimized system, and then used for testing 10 mating type isolates of Sporisorium scitamineum with different geographical origins and different hosts. A total of 86 bands were generated from the 10 SCoT primers, of which 58.67% was polymorphic and each primer generated 8.60 bands in average. UPGMA cluster analysis showed that 10 isolates could be divided into 3 groups with a coefficient of 0.75, and the genetic diversity has certain correlation with geographical origins. 