Serum alpha-fetoprotein (AFP), Lens culinaris agglutinin-reactive AFP (AFP-L3), and des-gamma-carboxyprothrombin (DCP) are useful biomarkers of hepatocellular carcinoma (HCC). However, associations among molecular characteristics and serum biomarkers are unclear. We analyzed RNA expression and DNA variant data from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) to examine their associations with serum biomarker levels and clinical data. From 371 TCGA-LIHC patients, we selected 91 seen at 3 institutions in Korea and the United States and measured AFP, AFP-L3, and DCP from preoperatively obtained serum. We conducted an integrative clinical and molecular analysis, focusing on biomarkers, and validated the findings with the remaining 280 patients in the TCGA-LIHC cohort. Patients were categorized into 4 subgroups: elevated AFP or AFP-L3 alone (↑AFP&L3), elevated DCP alone (↑DCP), elevation of all 3 biomarkers (↑All), and reference range values for all biomarkers (RR). CTNNB1 variants were frequently observed in ↑DCP patients (n = 7 [53.8%]) and RR patients (n = 10 [38.5%]), but ↑DCP patients with a CTNNB1 variant had worse survival than RR patients. TP53 sequence variants were associated with ↑AFP (n = 8 [30.8%]) and ↑DCP (n = 4 [30.8%]). The Wnt signaling pathway was activated in the ↑AFP&L3, whereas liver-related Wnt signaling was activated in the RR. TGF and VEGF signaling are activated in high AFP&L3, while dysregulated bile acid and fatty acid metabolism were dominant in ↑DCP. We validated these finding using the remainder of the TCGA-LIHC cohort and showed similar results to the test cohort. Serum AFP, AFP-L3, and DCP levels can help predict variants in the genetic profile.