Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide. Although the clinical success rate for the treatment of early-stage HCC has improved, the prognosis of advanced HCC remains poor owing to the high recurrence rate and the refractory nature of HCC for various anticancer drugs. A better understanding of the pathogenesis of HCC is therefore critically needed in order to treat HCC, including its genetic alterations. Next-generation sequencing (NGS) has provided an unbiased platform to systematically identify gene mutations and reveal the pathogenesis of various cancers. In the present study, a total of 118 samples (59 liver tissues including cancer and adjacent normal tissues) were sequenced using the AmpliSeq Hotspot Cancer Panel (version 2). The most common somatic mutations identified were tumor protein 53 (TP53; 35.6%) and β-catenin 1 (CTNNB1; 30.5%), and the most frequent variants of those genes were missense variants. In addition, somatic mutations including those in genes encoding colony-stimulating factor 1 receptor (5.1%), epidermal growth factor receptor (6.8%), RET proto-oncogene (3.4%), Erb-B2 receptor tyrosine kinase 4 (ERBB4; 1.7%) and serine/threonine kinase 11 (STK11, also known as liver kinase B1; 6.8%) were also identified at a low frequency in patients with HCC. A frameshift variant in STK11, a splice acceptor variant in TP53, a splice region variant in ERBB4 and a stop-gained variant in TP53 were also specifically determined. The most abundant alteration was a C:G>T:A transition (50%) and other transversions, i.e., C:G>G:C (19.6%), T:A>C:G (19.6%), C:G>A:T (12.5%), T:A>G:C (12.5%) and T:A>A:T (5.4%). This spectrum pattern differs from that in other solid tumors. TP53 mutations in the tumors at advanced stages were significantly more frequent compared with those in early-stage tumors. Additionally, age (<70 vs. ≥70 years) was significantly associated with CTNNB1 mutations. Using NGS, a number of novel gene mutations were identified in HCC, including established mutations and disproved mutations. The results of the present study offer new insight and improved understanding of the etiology and the development of HCC.