Guangxi province has the most abundant Oryza officinalis Wall germplasm resource in China. In this study, 1610 Oryza officinalis individuals which collected from all over Guangxi in situ sites, were used for genetic diversity analysis with 25 SSR markers. 181 polymorphic bands were detected, and the effective number of alleles (Ae) ranged from 1.0094 to 2.2674 and averaged at 1.3568. The expected heterozygosity (He) ranged from 0.0093 to 0.5591 and averaged at 0.2212. The Shannon-Weaver information index ranged from 0.0393-0.9296 and averaged at 0.3624. We confirmed that Wuzhou and Nanning are the genetic diversity center of Oryza officinalis in Guangxi and in China as well. Mantel correlation test was carried out to examine the correlation among cophenetic matrices, the results showed that more than 21 pairs of primer (no less than 10 pairs) would be the appropriate number for genetic diversity analysis of Oryza officinalis Wall populations.