Congenital heart disease (CHD) is one of the most common causes of neonatal mortality. The worldwide morbidity of CHD is 9.410‰, while the proportion of CHD patients who are reported to carry mutations in coding regions is < 50%. Enhancers play an important role in the spatio-temporal expression of target genes, and a lot of related variations are associated with CHD. There are conserved and nonconserved enhancers. It is common to study conserved enhancers in model organisms, while nonconserved enhancers lack systematic investigation and functional validation. Unfortunately, lots of CHD patients have no diagnosis even after whole exome sequencing (WES) analysis, while the significance of nonconserved regulatory regions, one of the potential pathogenic factors, is usually overlooked. Therefore, inthisstudy, to facilitate studies on the etiology of CHD, we present a systematic genome-wide analysis and functional validations of nonconserved human-specific enhancers (NoHEs).