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The ability to extract mitochondrial and nuclear DNA from ancient remains has enabled the study of ancient DNA, a legitimate field for over 20 years now. Recently, Y chromosome genotyping has begun to be applied to ancient DNA. The Y chromosome haplogroup in East Asia has since caught the attention of molecular anthropologists, as it is one of the most ethnic-related genetic markers of the region. In this paper, the Y chromosome haplogroup of DNA from ancient East Asians was examined, in order to genetically link them to modern populations. Fifty-six human remains were sampled from five archaeological sites, primarily along the Yangtze River. Strict criteria were followed to eliminate potential contamination. Five SNPs from the Y chromosome were successfully amplified from most of the samples, with at least 62.5% of the samples belonging to the O haplogroup, similar to the frequency for modern East Asian populations. A high frequency of O1 was found in Liangzhu Culture sites around the mouth of the Yangtze River, linking this culture to modern Austronesian and Daic populations. A rare haplogroup, O3d, was found at the Daxi site in the middle reaches of the Yangtze River, indicating that the Daxi people might be the ancestors of modern Hmong-Mien populations, which show only small traces of O3d today. Noticeable genetic segregation was observed among the prehistoric cultures, demonstrating the genetic foundation of the multiple origins of the Chinese Civilization. [ABSTRACT FROM AUTHOR]