Background: Ventricular septal defect (VSD) is the most common congenital cardiac abnormality in children and the second most common in adults. This study aimed to explore the potentially causative genes in VSD patients in the Chinese Tibetan population, and to provide a theoretical basis for the genetic mechanism of VSD. Methods: Peripheral venous blood was collected from 20 VSD subjects, and whole-genome DNA was extracted. High-throughput sequencing was performed on qualified DNA samples using whole-exome sequencing (WES) technology. After filtering, detecting, and annotating qualified data, single nucleotide variations (SNVs) and insertion-deletion (InDel) markers were analyzed, and data processing software such as GATK, SIFT, Polyphen, and MutationTaster were used for comparative evaluation and prediction of pathogenic deleterious variants associated with VSD. Results: A total of 4793 variant loci, including 4168 SNVs, 557 InDels and 68 unknown loci and 2566 variant genes were obtained from 20 VSD subjects through bioinformatics analysis. According to the screening of the prediction software and database, the occurrence of VSD was predicted to be associated with five inherited pathogenic gene mutations, all of which were missense mutations, including Conclusion: This study demonstrated that