In French Polynesia, the black-lip pearl oyster Pinctada margaritifera has been farmed to produce pearls since the 1980s, forming the basis of a major industry. The sustainability of this activity relies on spat collection in the lagoons. However, pearl oyster spat can be difficult to identify for the evaluation of stock variations. It is especially hard to distinguish Pinctada spp. larvae at a very early stage of development. In the present study, a whole- mount in situ hybridisation (ISH) technique was developed to allow the discrimination of larvae of closely-related pearl oyster species found in the French Polynesian atolls. Using specific ribosomal 16S-DNA sequence data, we were able to successfully differentiate between Pinctada margaritifera and Pinctada maculata larvae from 5 to 13 days old. This is the first description of a non-destructive method allowing bivalve larvae discrimination between species within this genus. The method allowed us to successfully identify P. margaritifera larvae in natural plankton samples. This result is a key step needed to develop monitoring of P. margaritifera larval distribution in French Polynesian lagoons, a procedure which will increase spat collection efficiency and ensure sustainable development of pearl oyster farming.