Focal nodular hyperplasia (FNH) is considered a benign tumor of the liver. However, the biologic nature and clonality status of FNH are not well established. We sought to determine the clonality and TP53 mutation status of FNH to better characterize the nature of FNH. We analyzed 15 cases of FNH from female patients who underwent surgical resection of their lesions. Genomic DNA was extracted from paraffin-embedded tissue sections using laser-capture microdissection and analyzed for X-chromosome inactivation status and TP53 mutations by direct DNA sequencing. Thirteen cases were informative for X-chromosome inactivation analysis. Of the 13 informative cases, 4 (31%) showed a nonrandom pattern of X-chromosome inactivation, consistent with monoclonal origin. No TP53 mutations were detected in any of the FNH cases. The clonality status was not associated with any clinicopathologic parameters such as age and lesion size. Our data indicate that a significant proportion of FNH lesions have a monoclonal origin, suggesting that they are neoplastic rather than reactive.