Background: Most cases of congenital heart disease (CHD) are sporadic and nonsyndromic, with poorly understood aetiology. Rare genetic variants have been found to affect the risk of sporadic, non-syndromic CHD, but individual studies to date are of only moderate sizes, and none to date has incorporated the ohnolog status in candidate gene studies. Ohnologs are genes retained from ancestral whole-genome duplications during evolution; multiple lines of evidence suggest ohnologs are over-represented among dosage-sensitive genes. Integration of large-scale data on rare variants was done with evolutionary information on ohnolog status to identify genetic loci predisposing to CHD. Methods: This was achieved by integrating publicly available data on copy number variation (CNV) in 4,634 non-syndromic CHD cases with in-house whole-exome sequencing (WES) data from 829 sporadic, non-syndromic patients with Tetralogy of Fallot (TOF). Those findings were placed in an evolutionary context by comparing the proportion of vertebrate ohnologs in CHD cases and controls. Further 63 non-TOF CHD samples undergone WES to identify likely deleterious variants. Subsequently, functional analysis of the strongest candidate, Slit3, was done by performing gene expression experiments and using mouse models to characterise in detail the impact of dysregulation of Slit3 in heart development. Results: Novel genetic loci in CHD cases were significantly enriched for ohnologs compared to the genome. This study has identified 54 novel candidate protein-coding genes supported by both: (i) CNV and TOF WES data; and (ii) ohnolog status. These 54 novel genetic loci had significantly more deleterious variants compared to the rest of the human genome in an independent non-TOF CHD cohort. Both Slit3 knockout and missense mouse models exhibit cardiac defects, where mouse embryos appear to have defects in neural crest cells in the absence of Slit3 during mouse embryonic development. Conclusions: This study has identified and also confirmed in a second independent CHD cohort, 54 novel CHD genetic loci. Ohnologs were shown for the first time to be overrepresented among CHD genes. Incorporation of evolutionary metrics may be useful in refining candidate genes emerging from large-scale genetic evaluations of CHD. Together with the functional experiments of Slit3, this study had provided novel insights of the role of Slit3 in cardiac development.