Animal studies have shown that knockout of the transforming growth factor beta-2 (TGFβ2) gene results in diverse cardiovascular malformations and that its unregulated expression is involved in the pathogenesis of heart defects. However, little information is available on the genetic and expression alternations of the TGFβ2 gene in children with congenital heart disease. This study investigated the genotype and expression of the TGFβ2 gene in children with congenital conotruncal defects (CTDs). The whole coding region of the TGFβ2 gene was sequenced in 400 children with CTD. The mRNA and protein expression of the TGFβ2 gene was further analyzed in the myocardial tissues of 37 children with CTD and 5 age-matched healthy children using real-time polymerase chain reaction and immunohistochemistry. No pathogenic mutations in the coding region of the TGFβ2 gene were shown by DNA sequencing except for a silent mutation (c.597T > C) in exon 4 of one patient. The TGFβ2 expression at either the mRNA or the protein level in the myocardial tissues did not differ significantly between the children with CTD and the children without heart defects. The results indicate that germline mutation of the TGFβ2 gene is not a common cause of CTD in humans and that the TGFβ2 expression level may be less critical in humans than in animals for the pathogenesis of CTD.