Introduction: Bicuspid aortic valve (BAV) is the most common congenital cardiovascular malformation. Individuals with BAV are at increased risk of developing aortic valve dysfunction, thoracic aortic aneurysm and dissection. In recent reports from familial and sporadic cases, genotypic variations of NOTCH1 gene in patients with BAV contribute to calcification and phenotypic variation. However, there is paucity of data on genotypic variations in Asian patients with BAV. This study provides data on targeted sequencing in search for novel NOTCH 1 gene mutations in a large number of Korean BAV patients. Methods: We performed a targeted mutational analysis of NOTCH1 gene using genomic DNA from 228 unrelated subjects. Denaturing high-performance liquid chromatography and DNA sequencing were performed. We focused on the 4 exons (11, 20, 25 and 29) in which mutations associated with BAV patients have been reported previously. Among 228 patients, the whole exon sequencing was performed in high risk patients who underwent valve replacement before the age of 40. The SNP sites, newly identified in high risk patients, were genotyped in remained patients. Results: Our analyses revealed 43 NOTCH1 sequence variants. 17 variants (11 novel mutation) are located within exon and 25 within intron (8 novel mutation). Among 17 heterozygote variants in exons, 12 variants are non-synonymous mutations, and 28% of the whole subjected were involved in these mutations. SNP at rs61751550 was associated with an odds ratio for valve failure before the age of 40 (odds ratio (OR) 29.1, 95% CI 2.9-292.8, p =0.0042). Newly identified E181K and N1156N SNPs were also significantly associated with early valve failure (OR 26.2, 95%CI: 1.1 to 662.5, p=0.047) Conclusion: This largest analysis involving targeted NOTCH1 gene in Korean BAV patients revealed novel mutations which were not described in western country. Three genetic markers at NOTCH1 gene may be useful in tailoring screening approaches in high-risk patients.