In this study, we aimed to investigate differential gene expression among spat of the pearl oyster Pinctada margaritifera with distinct growth phenotypes. Two groups of P. margaritifera spat belonging to the same F2 cohort were selected. Larvae were settled in controlled hatchery tanks using mussel rope collectors. After 5.5 months of rearing, the largest (n = 10) and smallest (n = 10) individuals, representing the head and the tail of the size distribution were selected as "Fast-growing" (hereafter referred to as F) and "Slow-growing" (hereafter referred to as S) phenotypes. Oysters were then dissected and their whole fresh tissues individually collected and preserved in RNAlaterat -80C until RNA extraction. Total RNA extraction was performed on the whole soft tissues of animals using TRIzolreagent (Invitrogen), following manufacturers recommendations. RNA integrity was assessed on a Bioanalyzer 2100 (Agilent Technologies, USA). Total RNA was dried in RNA-stable solution (Thermo Fisher Scientific) following manufacturers instructions and shipped at room temperature to McGill sequencing platform services (Montreal, Canada). RNA-seq libraries were generated using an Illumina TruSeq RNA Sample preparation kit according to manufacturers instructions (Illumina). RNA libraries were multiplexed (n = 10 individual libraries per sequencing lane) and sequenced on an Illumina HiSeq 4000 to produce 100-bp paired-end reads.