Abstract: Objective To explore the correlation between mutations in the promoter region of TBX1 gene and conotruncal heart defects. Methods A total of 621 children with conotruncal heart defects were recruited. Multiplex ligation-dependent probe amplification (MLPA) was used to detect the copy numbers of chromosomal region 22q11.2. Children with 22q11.2 deletion were excluded. Polymerase chain reaction amplification (PCR) and gene sequencing were applied to analyze promoter region of TBX1 (-2000..+1) in 605 children with conotruncal heart defects without 22q11.2 deletion and 588 healthy children. Bioinformatics software was used to predict and analyze the function of the variable loci. Results There were mutations in the promoter region of TBX1 gene in children with conotruncal heart defects, including 3 single nucleotide polymorphisms (SNP) sites and 7 rare loci. The incidence of mutation was 1.7%. The analysis of 7 rare loci by AliBaba 2.1 to showed that 3 of them may influence the combination of trans-acting factors and cis-acting elements of the promoter of TBX1 gene. Conclusion The mutation in the TBX1 promoter region may be related to the occurrence of conotruncal heart defects.