Abstract: Pteria penguin is one species of large pearl oysters and widely distribute in tropical and subtropical oceans. There are some wild populations distribute in Taiwan, Guangdong, Guangxi, and Hainan Island. As ideal pearl oyster producing high quality large hemispherical pearls and rare expensive round pearls, Pteria penguin has become a new cultivated species of pearl oyster in south China. To study the mechanism of the genetic diversity of different Pteria penguin populations, fluorescent amplified fragment length polymorphism (fAFLP) method was used to study the populations genetic diversity of Weizhou Island in Guangxi, Liusha Bay in Guangdong and Li'an in Hannan on three different geographical Pteria penguin respectively. fAFLP amplified ninety individuals from three different populations (thirty individuals per population) with seven pairs of primers. The result showed that every individual could be amplified clear and repeatable bands. The numbers of amplified loci per primer combination ranged from 100 to 163, and a total of 895 loci were detected in three populations, there were 865 polymorphic bands, the percentage of polymorphism of Guangxi Weizhou Island population, Guangdong Liusha Bay population and Hannan Li'an population were 70.73%, 63.13% and 66.82%. The Nei's gene diversity were 0.1634, 0.1558 and 0.1783, and Shannon's information index were 0.2635, 0.2474 and 0.2932, respectively. The genetic similarities were from 0.9722 to 0.9824 of three different populations. The genetic distance was from 0.0177 to 0.0282. UPGMA clustering chart was drawn according to genetic distance, but the results of Mantel test showed that there was no significant correlation between geographic distance and genetic distance of three Pteria penguin populations. Shannon's information index and molecular variance (AMOVA) analysis three populations diversity, the result indicated that three populations mainly came from different individuals within a population, among populations had 7.91% genetic variation and inner population had 92.09% genetic variation. The distribution of dominant gene frequency and gene flow Nm were analyzed, the result revealed that the three populations had same genetic structure, and there was clearly gene flow. The research result showed that Pteria penguin of Guangxi Weizhou island population, Guangdong Liusha Bay population and Hannan Li'an population had high percent of polymorphic loci, however, the populations did not exist significantly geographical differentiation. The result provided a reference evidence for breeding and establishment of protecting measures in Pteria penguin.