Background and Aims: Hepatocellular carcinoma (HCC) is highly malignant and prone to recur after surgical treatment. Differentiation between a true relapse of HCC and a second primary tumor is of clinical importance. However, no convenient method is currently available. Methods: Comparative genomic hybridization (CGH) was used to analyze 31 pairs of initial and recurrent HCC samples obtained from patients undergoing 2 consecutive surgeries. The resulting chromosomal aberration profiles were used as genomic fingerprints to determine tumor clonalities and their relationships. Results: Eleven recurrent tumors with high clonal relationship (CR) values (>0.95) were found to be relapsed HCCs, and 11 tumors with CR values close to 0 were found to be second primary HCCs. The other 9 paired samples had inconclusive CR values between 0.95 and 0.4. Two were confirmed by hepatitis B virus integration and X chromosome inactivation analysis to be de novo cancers (CR values, 0.35 and 0.23, respectively). Initial HCCs that subsequently relapsed accumulated more chromosomal aberration events than those that developed de novo HCC (mean, 16.1 ± 4.5 vs. 5.4 ± 4.8 events; P < 0.01). Also, they more frequently showed gains on chromosome arms 3q, 6p, 8q, and 17q and losses on 4q and 16p. Conclusions: CGH is useful for chromosomal aberration study and tumor clonality analysis. More and characteristic genomic changes in the initial HCC suggest that subsequent tumor recurrence is a true relapse.