Abstract: Sugarcane smut caused by Ustilago scitaminea is a worldwide disease and causes serious damage to sugarcane. Research on the pathogen of sugarcane smut is aid to sugarcane breeding resistant to smut. With the conservative regions and variable regions in rDNA gene sequences, the analysis of rDNA sequences is very efficient in research of fungi systemic development, classification identification and molecular detection. The method of single spore isolation is occupied in this experiments. Single spore was isolated from smut whip in infected plant of smut high resistant sugarcane variety of NCo376, and then its mycelia were prepared and used for isolation of genomic DNA. The rDNA analysis of the fungus will increase understanding of it. The universal primers of ITS1 and ITS4 for fungi were used in amplification of genomic rDNA sequences. A target fragment was obtained by ITS-PCR. Then, the fragment is cloned into the vector of pMD18-T before sequencing. The result of sequencing showed that the fragment is 692 bp in length. The results of Blast in Genbank and the analysis of fungal rDNA systemic development show that the relation between this fragment to the fungi of Sporisorium is closest, while the relation between it to the fungi of Ustilago is much far. It suggests that the name of Ustilago scitaminea for sugarcane smut isn’t consistent to the result of rDNA systemic development analysis. It still needs more experiments to support this hypothesis.