Background: Cultured pearls are unique gems produced by living organisms, mainly molluscs of the Pinctada genus, through the biomineralization properties of pearl sac tissue. Improvement of P. margaritifera pearl quality is one of the biggest challenges that Polynesian research has faced to date. To achieve this goal, a better understanding of the complex mechanisms related to nacre and pearl formation is essential and can now be approached through the use of massive parallel sequencing technologies. The aim of this study was to use RNA-seq to compare whole transcriptome expression of pearl sacs that had producing pearls with high and low quality. For this purpose, a comprehensive reference transcriptome of P. margaritifera was built based on multi-tissue sampling (mantle, gonad, whole animal), including different living stages (juvenile, adults) and phenotypes (colour morphotypes, sex). Results: Strikingly, few genes were found to be up-regulated for high quality pearls (n = 16) compared to the up-regulated genes in low quality pearls (n = 246). Biomineralization genes up-regulated in low quality pearls were specific to prismatic and prism-nacre layers. Alternative splicing was further identified in several key biomineralization genes based on a recent P. margaritifera draft genome. Conclusion: This study lifts the veil on the multi-level regulation of biomineralization genes associated with pearl quality determination.