Brugada syndrome (BrS) is one of the ion channelopathies associated with sudden cardiac death (SCD). The most common BrS-associated gene (SCN5A) only accounts for approximately 20-25% of BrS patients. This study aims to identify novel mutations across human ion channels in non-familial BrS patients without SCN5A variants through disease-targeted sequencing. We performed disease-targeted multi-gene sequencing across 133 human ion channel genes and 12 reported BrS-associated genes in 15 unrelated, non-familial BrS patients without SCN5A variants. Candidate variants were validated by mass spectrometry and Sanger sequencing. Five de novo mutations were identified in four genes (SCNN1A, KCNJ16, KCNB2, and KCNT1) in three BrS patients (20%). Two of the three patients presented SCD and one had syncope. Interestingly, the two patients presented with SCD had compound mutations (SCNN1A:Arg350Gln and KCNB2:Glu522Lys; SCNN1A:Arg597* and KCNJ16:Ser261Gly). Importantly, two SCNN1A mutations were identified from different families. The KCNT1:Arg1106Gln mutation was identified in a patient with syncope. Bioinformatics algorithms predicted severe functional interruptions in these four mutation loci, suggesting their pivotal roles in BrS. This study identified four novel BrS-associated genes and indicated the effectiveness of this disease-targeted sequencing across ion channel genes for non-familial BrS patients without SCN5A variants.