Aldehyde dehydrogenase isoforms, ALDH1A1 and ALDH3A1, are associated with poor clinical outcome and resistance to chemotherapy in a wide variety of human malignancies. So far, their expression and prognostic significance in hepatocellular carcinoma (HCC) remains unknown. The aim of our study was to investigate their expression in HCC, and to correlate this to clinical, pathological and molecular features. ALDH1A1 and ALDH3A1 expression was first evaluated by microarray analysis in a series of 60 HCCs and five tumour-free liver tissue samples. Our findings related to ALDH3A1 were further validated by immunohistochemistry in a series of 81 HCCs and 23 hepatocellular adenomas (HCA). Microarray analysis showed no difference in ALDH1A1 expression between HCCs and tumour-free liver tissue. In contrast, ALDH3A1 was strongly upregulated in a subset of HCCs characterised by activation of the Wnt/ß-catenin pathway and CTNNB1 mutations. Using immunohistochemistry, we confirmed that high ALDH3A1 expression is associated with nuclear staining for ß-catenin and strong homogeneous staining for glutamine synthetase, two classical Wnt/ß-catenin pathway activation markers. Consistent with this finding, in tumour-free liver tissue, ALDH3A1 expression was observed in centrilobular hepatocytes, in which the Wnt/ß-catenin pathway is known to be physiologically activated. We also observed higher ALDH3A1 expression in CTNNB1-mutated HCA when compared with other subtypes. No correlation between ALDH3A1 expression and patient survival or tumour recurrence was observed.In conclusion, ALDH3A1 is a marker of activation of the Wnt/ß-catenin pathway in HCC, HCA and tumour-free liver tissue. Further studies may help to elucidate the potential role of ALDH3A1 in HCC development and resistance to chemotherapy.