The French Polynesian islands are internationally known for their black pearls, produced by culture of the black lipped pearl oyster Pinctada margaritifera. The ongoing development of hatcheries for P. margaritifera in French Polynesia poses new challenges for the industry, particularly regarding the maintenance of genetic diversity in the hatchery stocks. This emphasizes the necessity to characterize the genetic diversity and differentiation within natural and exploited populations, to carefully select putative parental populations. The present study aimed at validating the phylogenetic status and investigating genetic attributes of populations from the only two non-exploited archipelagos of French Polynesia, the Marquesas archipelago, and the Australes archipelago, never analysed before. We found that individuals from both archipelagos belonged to P. margaritifera species. However, while the Australes population was genetically similar to non-exploited populations of the Tuamotu, the Marquesas populations were highly differentiated from the rest of the populations. This differentiation cannot not be only attributed to geographic distance and aquaculture status, but likely to hydrodynamic barriers allowing vicariant events to take place. our results add up to other studies describing the Marquesas archipelago as a hotspot for biodiversity and differentiation, with some of the highest levels of endemism and vicariance found among marine species worldwide and provide precious information on available genetic resources for the implementation of P. margaritifera selective breeding and its genetic conservation in french polynesia.