ABSTRACT. Sugarcane smut caused by the fungus Sporisorium scitamineum is a worldwide disease and also one of the most prevalent diseases in sugarcane production in mainland China. To study molecular variation in S. scitamineum, 23 S. scitamineum isolates from the 6 primary sugarcane production areas in mainland, China (Guangxi, Yunnan, Guangdong, Hainan, Fujian, and Jiangxi Provinces), were assessed using internal transcribed spacer (ITS) methods. The results of ITS sequence analysis showed that the organisms can be defined at the genus level, including Ustilago and Sporisorium, and can also differentiate between closely related species. This method was not suitable for phylogenetic relationship analysis of different S. scitamineum isolates and could not provide support regarding their race ascription at the molecular level. The results of the present study will be useful for studies examining the molecular diversity of S. scita-7895 Molecular variation of Sporisorium scitamineum ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (3): 7894-7909 (2015) mineum and for establishing a genetic foundation for their pathogenicity differentiation and new race detection. In addition, our results can provide useful information for the pathogen selection principle in sugarcane smut resistance breeding and variety distribution.