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Saccharum spontaneum is a wild sugarcane species that is native to and widely distributed in China. It has been extensively used in sugarcane breeding programs, and is being tested for the development of bioenergy cultivars. In order to provide basic information for the exploitation of this species, we analyzed genetic variation among and within native S. spontaneum populations collected from Sichuan, China. Eighty plants from nine native populations were sampled. Twenty-one sequence-related amplified polymorphism primer pairs generated 235 clearly scorable bands, of which 185 were polymorphic (78.7%). Nei's genetic diversity was 0.2801 and Shannon's information index was 0.4155 across the populations. Genetic diversity parameters, G(ST) value (0.2088) and N(m) value (1.8944), showed that the genetic variation within populations was greater than that among populations. In the cluster analysis, one major grouping was formed by populations from Ya'an and another one by populations from Sichuan basin; a population from Baoxing formed a single cluster. In order to fully comprehend the genetic diversity of cold-tolerant local germplasm in this species, germplasm should be collected from the heterogeneous environments along the northern regions of this species' distribution. The germplasm that we collected should be a valuable resource for Saccharum breeding.