Heart development is complex and requires the sequential and timely interplay of regulatory master proteins, notably several transcription factors. Germline mutations in the human transcription factor genes such as NKX2-5, TBX5 and GATA4 are associated with cardiac anomalies. Familial cases so far studied have different mutations and no mutation can be associated with a specific clinical phenotype. Many cases of CHD come from unaffected family members. We searched for sequence alterations in cardiac specific transcription factor genes that would lead to loss-of-function of the protein in 68 malformed hearts from the Leipzig heart collection. By direct DNA sequencing, we obtained mutations in several transcription factors e.g. NKX2-5, TBX5 and GATA4 in the diseased heart tissues, which were mainly absent in normal heart tissues of the same CHD patients. We also observed multiple mutations in patients, multiple haplotypes, and detection of mutations in Down syndrome patients, all suggesting somatic origin of mutations and genomic instability in the diseased cardiac tissues of patients with CHD. Somatic mutations may provide an alternate mechanism of disease.