Congenital heart disease (CHD) is a genetically heterogeneous disease. Targeted next-generation sequencing (NGS) offers a unique opportunity to sequence multiple genes at lower cost and effort compared to Sanger sequencing. We tested a targeted NGS of a specific gene panel in a relatively large population of non-syndromic CHD patients. The patient cohort comprised 68 CHD patients (45 males; 8.3 ± 1.7 years). Amplicon libraries for 16 CHD-strictly related genes were generated using a TruSeq^® Custom Amplicon kit (Illumina, CA) and sequenced using the Illumina MiSeq platform. Sequence data were processed through the MiSeq Reporter and wANNOVAR softwares. After applying stringent filtering criteria, 20 missense variants in 9 genes were predicted to be damaging and were validated by Sanger sequencing with 100% concordance. Fourteen variants were present in public databases with very rare allele frequency, of which four variants (p.Arg25Cys in NKX2-5, p.Val763Ile in ZFPM2, p.Arg1398Gln and Gly1826Asp in MYH6) have been previously linked to CHD or cardiomyopathy. The remaining six variants in four genes (GATA4, NKX2-5, NOTCH1, TBX1) were novel mutations, currently not found in public databases, and absent in 200 control alleles of healthy subjects. Four patients (5.8%) carried two missense variants (1 compound heterozygote in the same gene and 3 double heterozygotes in different genes), with possibly synergistic deleterious effects. Targeted NGS is a powerful and efficient tool to detect DNA sequence variants in multiple genes, providing the opportunity for discovery of the co-occurrence of two or more missense rare variants.