We used a RNA-seq approach to determine the effect of 3,5-di-iodothyronine (T2) and 3,5,3'-tri-iodothyronine (T3) exogenous treatment on the transcriptome of tilapia (Oreochromis niloticus) liver, cerebellum, and thalamus-pituitary. We identified a total of 169, 154 and 2863 genes that were Thyroid hormone-regulated (FDR < 0.05) in tilapia cerebellum, thalamus-pituitary and liver, respectively. Among those, 130, 96 and 349 genes were uniquely regulated by T3, whereas 22, 40 and 929 were only regulated by T2 in the cerebellum, thalamus-pituitary and liver. Overall design: Juvenile tilapia were treated by immersion protocol using these conditions: 25 nM of T2 or T3 for 12 h of exposure (n = 2 individuals for liver samples and 2 biological replicates of a pool of 4 individuals for the brain regions samples). Transcriptome sequencing was conducted using Genome Analyzer GAIIx (Illumina). A configuration for pair-end reads with a 72 bp read length was used.