Thyroid hormones (THs) play a key role in larval development, growth and metamorphosis in flatfish. Their genomic effects are mediated by thyroid hormone receptors (TRs). In this study, cDNAs encoding for TRalphaA, TRalphaB, and TRbeta have been sequenced in Senegalese sole (Soleasenegalensis). Main domains and conserved motifs were identified. Also, a truncated TRalphaB isoform (referred to as TRalphaBtr) and a spliced TRbeta variant (referred to as TRbetav) were detected. A phylogenetic analysis grouped both TRalpha and TRbeta genes into two separate clusters with their fish and mammalian counterparts. Expression profiles during larval development and in juvenile tissues were analyzed using a real-time PCR approach. In juvenile fish, TRalphaA, TRalphaB, TRbetav, and TRbeta showed distinct transcript levels in tissues. During metamorphosis, only TRbetav and TRbeta modified their mRNA levels in a similar way to the T4 contents. To evaluate the possible regulation of TRs by their cognate ligand T4 during sole metamorphosis, larvae were exposed to the goitrogen thiourea (TU). TRbeta transcripts decreased significantly at 11 and 15 days after treatment. Moreover, adding exogenous T4 hormone to TU-treated larvae restored the steady-state levels or even increased TRbeta and TRbetav mRNAs with respect to the untreated control. Overall, these results demonstrate that TRbeta transcription is up-regulated by THs playing a major role during metamorphosis in Senegalese sole.