This study aims to identify the genetic and epigenetic determinants of congenital heart defect (CHD) in Down syndrome (DS). We hypothesized that trisomy 21 in concert with additional genetic and epigenetic variations contribute to the risk of CHD in DS. Association studies were performed in samples from DS with and without CHD to identify genome-wide SNPs and chromosome 21 specific CNVs associated with CHD. The results revealed two SNPs and two CNVs on chromosome 21 that may modify the CHD risk in DS. Furthermore, a monozygotic twin approach was used to elucidate the role of trisomy 21 in DNA methylation modifications. Reduced representation bisulphite sequencing (RRBS) revealed differentially methylated regions in promoters of genes involved in embryonic organ morphogenesis relevant to DS phenotypes including CHD. In summary, the results of this work support a multifactorial model for development of CHD in DS that includes trisomy 21, SNPs, CNVs, and DNA methylation modifications.