Emperor CAO Cao (155AD-220AD) is one of the most famous persons in Chinese history that had changed the history of East Asia. He claimed to be a descendant of Marquis CAO Can and therefore was of aristocratic ancestry. However, this claim has been suspected for around 1800 years. Here, we collected some present clans with full records of 70-100 generations claimed to be descendants of CAO Cao or CAO Can, and validated them by comparing their Y chromosomes. Haplotype O2-M268 is the only one that is enriched significantly in the Emperor's claimed descendant clans (P=9.323 × 10(-5), odds ratio=12.72) and, therefore, is most likely to be that of the Emperor. Moreover, our analysis showed that the Y chromosome haplotype of the Emperor is different from that of the Marquis (Haplotype O3-002611). Therefore, Emperor CAO Cao's claim was not supported by genetic evidence. This study offers a successful showcase of the utility of genetics in studying the ancient history.