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Yunnan Province of China is one of the important centers for origin and evolution of cultivated rice worldwide. Wild rice is the ancestor of the cultivated rice. Many elite traits of wild rice have widened the genetic basis in cultivated rice. However, many populations of wild rice species have disappeared in the past few years. Therefore, the current status of wild rice resources should be updated and the genetic diversity of wild rice species should be examined for further germplasm preservation and utilization. Our investigations showed that the number of natural wild rice populations declined sharply in Yunnan Province during the past few years due to various reasons. Fortunately, one population of Oryza rufipogon, three of O. officinalis and ten of O. granulata have been newly found in different ecological sites, which were confirmed by inter-simple sequence repeat (ISSR) marker analysis in this study. ISSR analysis and investigation of some important traits of nutritional values indicated that the genetic diversity of the currently existing wild rice resources in Yunnan is still rich. The demonstration of genetic diversity of wild rice by a combined use of geographical distribution, morphological traits, nutrition contents and ISSR markers would be helpful for the conservation and exploration of these important wild rice resources.