Twenty-five populations of Oryza rufipogon from China and 144 cultivars of Oryza sativa were selected for this study. Based on the DNA fragment of Ehd1-4 and subspecies-specific sequence-tagged site markers in different chromosomes, intraspecific differentiation in O. rufipogon from China was analyzed. The introgression from O. sativa to O. rufipogon was also analyzed based on simple sequence repeat markers. The results revealed that the DNA fragment of Ehd1-4 could distinguish the O. sativa subspecies japonica and indica. Furthermore, although significant indica-japonica differentiation did not occur in most O. rufipogon populations from China, O. rufipogon varieties from Hainan Island and from the mainland of China showed differentiation tendencies. Japonica-like O. rufipogon varieties were predominant in Mainland China. However, more indica-like O. rufipogon varieties were found in Hainan Island. Finally, although cultivar-specific alleles were found in most of the O. rufipogon varieties from Hainan Island and Guangdong Province, some varieties remain pure and non-introgressive.