Background: The NKX2 gene family is made up of core transcription factors that are involved in the morphogenesis of the vertebrate heart. NKx2-5 plays a pivotal role in mouse cardiogenesis, and mutations in NKx2-5 result in an abnormal structure and function of the heart, including atrial septal defect and cardiac electrophysiological abnormalities. Material/Methods: To investigate the genetic variation of NKX2-5 in Chinese patients with sporadic atrial septal defect, we sequenced the full length of the NKX2-5 gene in the participants of the study. Four hundred thirty-nine patients and 567 healthy unrelated individuals were recruited. Genomic DNA was extracted from the peripheral blood leukocytes of the participants. DNA samples from the participants were amplified by multiplex PCR and sequenced on an Illumina HiSeq platform. Variations were detected by comparison with a standard reference genome and annotation with a variant effect predictor. Results: Thirty variations were detected in Chinese patients with sporadic atrial septal defect, and 6 single nucleotide polymorphisms (SNPs) had a frequency greater than 1%. Among the 30 variations, the SNPs rs2277923 and rs3729753 were extremely prominent, with a high frequency and odds ratio in patients. Conclusions: Single nucleotide variations are the prominent genetic variations of NKX2-5 in Chinese patients with sporadic atrial septal defect. The SNPs rs2277923 and rs3729753 are prominent single nucleotide variations (SNVs) in Chinese patients with sporadic atrial septal defect. © 2019, International Scientific Information, Inc.. All rights reserved.