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The present study was conducted to assess the molecular characterization and genetic diversity amongst natural populations of Myrica rubra in Guangxi Zhuang Autonomous Region, China, thus to provide scientific evidence for germplasm conservation and exploitation. Using ISSR (inter-simple sequence repeats) markers, the level of genetic variation and the molecular characterization of 10 natural populations of M. rubra, originated from Guangxi Zhuang Autonomous Region in China, were performed. Based on 11 primers, 123 clear and reproducible DNA fragments were generated, of which 95 (77.24%) were polymorphic. The average value of Nei's gene diversity (He) was 0.268. The coefficient of genetic differentiation (Gst) was 0.341, revealing that 34.1% of the total molecular variance existed among populations. The Mantel statistical testing showed that the genetic distance was correlated to the geographic distance,but the correlation was not significant. Ten populations were divided into two big clusters according to unweighted pair group method with arithmetic mean (UPGMA) analysis. One consisted of populations of Rongxian (RX), Hepu (HP),Liangqing (LQ), Marshan (MS), Lingshan (LS) and Shansi (SS), which originated from the southern Guangxi, while the other was composed of Guanyang (GY) and Lingui (LG) populations of northern Guangxi, Huanjiang (HJ) populations of northwestern Guangxi and Shanglin (SL) populations of southern Guangxi. The level of genetic variation in wild M. rubra population distributed in Guangxi is high. Gene drift within the population was responsible for genetic variation in wild M. rubra in Guangxi, and the effect of the genetic flow among inter-populations was not significant. Classification of wild M. rubra populations was correlated to climate and environment. The molecular characterization and diversity assessment of M. rubra is of immense value for planning conservation of its genetic resources and their exploitation for further studies.