<b><i>Background:</i></b> Congenital primary hypothyroidism (CH) is the most common endocrine disorder in neonates. <b><i>Methods:</i></b> To identify novel genes, we performed whole exome sequencing (WES) in 6 patients with CH due to thyroid dysgenesis (TD). The potential effects of the most relevant variants were analyzed using in silico prediction tools. The most promising candidate gene, transient receptor potential channel 4-associated protein (<i>TRPC4AP</i>), was sequenced in 179 further patients with TD. Expression of <i>TRPC4AP</i> in human thyroid was investigated using RT-PCR. <i>Trpc4ap</i>- functional analysis was performed in <i>Xenopus laevis</i> using Morpholino (MO) antisense oligomers. <b><i>Results:</i></b> WES identified a likely damaging mutation in <i>TRPC4AP</i> leading to a de novo stop codon p.Q552*. Targeted sequencing of <i>TRPC4AP</i> demonstrated gene variants with predicted damaging potential in 5 patients resulting each in an amino acid exchange (p.P706S, p.F729L, p.S777C, and p.N229S). We demonstrated that <i>TRPC4AP</i> is expressed in human thyroid gland tissue. Using <i>Xenopus laevis</i>, we showed that the volume of the tadpole thyroid anlage was reduced by 20% in <i>Trpc4ap</i> MO knockdowns compared to controls and by 41% in “Clustered Regularly Interspaced Short Palindromic Repeats”/Cas9-mediated gene knockout experiments. <b><i>Discussion:</i></b> A recognized interaction of TRPC4AP and the NF-kappa-B-essential-modulator encoded by <i>IKBKG</i> gene was identified by IPA analysis. IKBKG plays a role in activation of the NF-κB-signaling pathway and regulates genes involved in proliferation and survival of thyrocytes and expression of key enzymes of thyroid hormone synthesis. <b><i>Conclusion:</i></b> <i>TRPC4AP</i> was identified as a novel candidate gene in TD, but further studies are needed to validate its role in thyroid function.