Saccharum spontaneum L. is the most important wild resource in sugarcane breeding. It is necessary to identify and collect these wiId germplasm for a better understanding the genetic background of Saccharum spontaneum L.. In this study, 11 original clones which had never been collected from Yunnan province as well as 14 control clones which have a better polymorphism from national nursery of sugarcane germ were amplified with 10 SSR primers. As a result, a total of 233 bands were acquired. Out of these bands, 207 (89%) were polymorphic bands and 14 were specific bands for the new collected clones with contrast to the control materials. The new collected materials could not be separated from the control by the genetic similarity coefficients and UPGMA cluster analysis. The 25 clones were divided into three groups at 0.64 of genetic similarity coefficient: the first group consists of Longmen, River Village Saccharum spontaneum L. and Fujian Xianyou No.1; the second group was clustered by 19 materials from inland areas, including 9 new gathering resources: Shanggang, Tala, Anle, Menggen, Mangmei, Hehai, Huiluo, Lila and Manheng Saccharum spontaneum L., and 10 control from Yunnan, Sichuan, Vietnam, Laos and Thailand area; the third group includes three control materials, Hainan No.1, Hainan 92-2 and Huazhou of Guangdong S.spontaneum L.. The second group was divided into 3 sub-groups at 0.654 of genetic similarity coefficient. These results indicated that the 11 new collections of S.spontaneum L. showed abundant genetic diversity in comparison with the control. Therefore, it supposed that there were striking genetic differences among these materials derived from the distinctive geographical distribution and climatic circumstances of Yunnan province.