To estimate genetic diversity of Oryza rufipogon Griff. in Southeast region of Guangxi in China, a total of 301 individuals form 8 natural populations in Hezhou City, Fangchenggang City and Chongzuo City were analyzed using 25 microsatellites loci. A moderate to high level of genetic diversity was observed at population levels with the mean number of alleles (A) ranged from 2.0400 to 5.2000 (overall 10.2400). The effective number of alleles (Ae) ranged from 1.5337 to 2 .9921 (overall 5 .0221) . The mean expected heterozy-gosity (He) ranged from 0.2838 to 0.5840 (overall 0.7641). The observed heterozygosity (Ho) ranged from 0.2150 to 0.5753 (overall 0.4840). An estimate of the outcrossing rate (t = 0.4570) suggests that the species has a typical mixed-mating system. Furthermore, a moderate genetic differentiation ( Fst = 0.3459) was found among the populations studied, suggesting that 35.49% genetic variation existed among the populations. Moreover, microsatellite showed less deviation from Hardy-Weinberg expectation within and among populations (Fis = 0.2680, Fit = 0.4817) and a slight deficiency of heterozygotes. Eventually together with the pattern of genetic differentiation of natural populations of Oryza rufipogon in Southeast region of Guangxi, it could be suggested that the population QT, YJ, TJ be given priority for in-situ conservation.