A general south-north genetic divergence has been observed among Han Chinese in previous studies. However, these studies, especially those on mitochondrial DNA (mtDNA), are based either on partial mtDNA sequences or limited samples. Given that Han Chinese comprise the world's largest population and reside around the world, whether the north-south divergence can be observed after all regional populations are considered remains unknown. Moreover, factors involved in shaping the genetic landscape of Han Chinese needs further investigations. In this study, we dissected the mtDNA landscape of Han Chinese by studying 4,004 mtDNA haplogroup-defining variants in 21,668 HanChinese samples from virtually all provinces in China. Our results confirmed the genetic divergence between southern and northern Hanpopulations. However, we found a significant genetic divergence among populations from the three main river systems, i.e., the Yangtze, the Yellow and the Zhujiang (Pearl) rivers, which largely attributed to the prevalent distribution of haplogroups D4, B4 and M7 in these rivervalleys. Further analyses based on 4,986 mitogenomes, including 218 newly generated sequences, indicated that this divergence was already established during the early Holocene and may have resulted from population expansion facilitated by ancient agricultures along these rivers. These results imply that the maternal gene pools of the contemporary Han populations have retained the genetic imprint of early Neolithic farmers from different river basins, or that river valleys represented relative migration barriers that facilitated genetic differentiation, thus highlighting the importance of the three ancient agricultures in shaping the genetic landscape of the Han Chinese