The TBX5 transcription factor plays an important role during morphogenesis and development of the heart. Mutations in this gene often lead to Holt-Oram syndrome (HOS). This study identified mutations in patients with non-syndromic congenital heart defects (CHD). Screening for mutations TBX5 gene in non-syndromic CHD, including 100 patients with a septal defect and 50 healthy subjects as controls were performed by the technique of high-resolution melt (HRM). Exons were sequenced for samples that showed HRM curve differences compared to controls. Structural stability and pathogenic potential of mutated protein were evaluated by bioinformatics analysis. HRM curve analysis showed that the curves of three samples deviated from the curves of controls. Sequencing showed three heterozygous missense mutations including two novel mutations NM_000192.3:c.44T>G, (p.L15Q), NM_000192.3c.629C>G (p.A210G) and a known mutation NM_000192.3:c.331G>T (p.D111Y). The PolyPhen-2 software predicted the p.D111Y and p.A210G substitutions to be disease-causing and p.L15Q as possibly benign, while protein structural stability analysis by MUpro and DynaMut suggested that these mutations reduce stability and increase the flexibility of the protein. This study presents two novel missense mutations within the TBX5 gene that may be causal for non-syndrome CHD.