Oryza rufipogon Griff. is the most agriculturally important but seriously endangered wild rice species. To better estimate how genetic structure can be used to obtained a conservation perspective of the species, genetic variability at six polymorphic microsatellite DNA loci was examined. High levels of genetic variability were detected at six loci in 1245 individuals of 47 natural populations covering most of the species’ range in China (overall RS = 3.0740, HO = 0.2290, HS = 0.6700). Partitioning of genetic variability (FST = 0.246) showed that most microsatellite variation was distributed within populations. Significant departures from Hardy–Weinberg expectations and very strong linkage disequilibrium indicate a high degree of inbreeding in the species and severe subdivision within populations. A mean Nm value of 0.7662 suggested a limited gene flow among the assayed populations. Our study suggests that conservation and restoration genetics should focus in particular on the maintenance of historically significant processes such as high levels of outbreeding and gene flow and large effective population size in the species.