It is often difficult to distinguish hepatocellular adenoma (HCA) from well-differentiated hepatocellular carcinoma (WDHCC) when limited tissue from a needle biopsy is evaluated. The aim of this study was to identify gene expression patterns that can distinguish HCA from WDHCC, with the ultimate goal of discovering novel diagnostic markers. Gene expression profile analysis was performed using Affymetrix U133Plus2 GeneChip microarrays on RNA isolated from frozen tissue of 6 HCA and 8 WDHCC specimens. Statistical analysis of microarray data identified 63 genes whose expression levels were significantly different between HCA and WDHCC. These included 57 genes overexpressed by HCA and 6 overexpressed by WDHCC. Eight genes were chosen for further analysis by quantitative RT-PCR on RNA derived from archived, paraffin-embedded tissue blocks of an independent validation set comprising 9 HCAs and 9 HCCs. Seven of the 8 genes demonstrated average expression differences between HCA and HCC that were concordant with the microarray findings, and their expression pattern correctly classified the 18 tumors into HCA and HCC using unsupervised clustering analysis. Furthermore, immunohistochemical staining performed on a third, independent set of 27 HCAs and 33 HCCs confirmed the expression differences at protein levels for 5 of the genes. Taken together, our data demonstrate significant molecular differences between HCA and WDHCC, despite their morphologic similarity. More importantly, we have identified a unique set of genes whose expression pattern can discriminate between these two types of hepatocellular neoplasms, suggesting the possibility of future development of ancillary molecular and immunohistochemical diagnostic methods.