Twenty-four SSR loci which were distributed evenly among the genome of cultivated rice were selected to assess the genetic diversity of 343 Oryza rufipogon individuals from 12 populations along the Haojiang River, Wuxuan County, Guangxi Zhuang Autonomous Region in China. The results indicated that: (i) Abundant genetic diversity existed: among 206 detected alleles, the percentage of polymorphic loci (p) ranged from 79% to 100% with the mean of 92.36%; the number of alleles per locus (Al) ranged from 3 to 19 with the average of 8.7083; the number of effective alleles (Ae) ranged from 1.2300 to 11.4195 with the mean of 3.7117; the Shannon's information index (I) ranged from 0.5540 to 1.1138 with the overall of 1.3692. (ii) High genetic differentiation among populations and gene flow were detected (Gst=0.2659; Nm=0.6901), suggesting that 26.59% genetic variation existed between the populations. (iii) Many rare alleles were detected. In 206 alleles, 65 were existed only in one or two populations, 12 of which existed only in population B. (iv) The results of cluster analysis and principle coordinates analysis (PCO) showed that population A and B from the downstream were close; population C from the middle was unique and became a cluster singly; the population D, E, and G from the middle were close; the population H and I from the middle and K, L from the upstream were close. It could be suggested that the populations from the middle and downstream of the Haojiang River, especially population B, D and H should be given priority to be conserved in-situ.