Objectives To diagnose undiagnosed heritable thoracic aortic disease (HTAD) using targeted next-generation sequencing technology on human aorta tissue obtained from a single-centre biobank.Methods From May 2016 to November 2021, 622 patients undergoing surgical repair for thoracic aortic aneurysm or acute aortic syndrome donated blood or tissue samples to the research biobank. A total of 134 aortic tissue samples were retrieved from patients suspected of HTAD. The inclusion criteria were (1) family history, (2) age <= 45, (3) annuloaortic ectasia, (4) bicuspid aortic valve, (5) patent ductus arteriosus, (6) polycystic kidney disease, or (7) patients previously classified as variant of uncertain significance by germline sequencing (n = 17). Exclusion criteria included patients previously classified as likely pathogenic or pathogenic by germline sequencing. The targeted panel included 96 genes.Results A total of 29 variants were identified including FBN1, MYH11, COL11A1, SMAD3, SMAD6, ACTA2, COL3A1, FLNA, PKD2, THSD4, ACVRL1, and FBN2. A total of 27 patients (20.1%) had variants with 2 patients having digenic variants. Clinical data were combined with genetic data, and finally, 17 patients (12.7%) were additionally diagnosed with HTAD.Conclusions Genomic data analysis of human aortic tissue allowed for additional diagnoses of undiagnosed HTAD, contributing to resolving discrepancies between clinical and genomic data. Considering the surgical treatment of HTAD, human aortic tissue can be a valuable resource for genomic analysis. Thoracic aortic aneurysm (TAA), characterized by progressive dilatation of the thoracic aorta, may culminate in acute aortic syndrome (AAS), including dissection or rupture.