The Han Chinese is the largest single ethnic group in the world, consisting of ten Chinese branches. With the exception of the Pinghua branch, the genetic structure of this group has been studied extensively, and Y chromosome and mitochondrial (mt)DNA data have demonstrated a coherent genetic structure of all Han Chinese. It is therefore believed that the Pinghua branch, being members of an old branch of the Han Chinese, despite being scattered in and around Guangxi Province where members of the Daic and Hmong-Mien are more prevalent than Han Chinese, is no exception. We have studied 470 individual samples (including 195 males) from Pinghua populations and other ethnic groups (Zhuang, Kam, Mulam, Laka, and Mien) from six areas (Hezhou, Fuchuan, Luocheng, Jinxiu, Sanjiang, and Wuxuan) in the north of the Guangxi Zhuang Autonomous Region of China. Both mtDNA and the Y chromosomes were typed in these samples. High frequencies of the Y chromosome haplogroups O2a* and O*, which always present at a high frequency among the populations of the southern minorities, were found in Pinghua populations. Only Pinghua populations in Luocheng and Jinxiu maintain the Han frequent haplogroup O3a5a. mtDNA lineages B4a, B5a, M*, F1a, M7b1, and N* were found in Pinghua populations, exhibiting a pattern similar to the neighboring indigenous populations, especially the Daic populations. Cluster analyses (dendrograms, principal component analyses, and networks) of Pinghua populations, the other Han branches, and other ethnic groups in East Asia indicated that Pinghua populations are much closer to the southern minorities than to the other Han branches. Admixture analyses confirmed this result. In conclusion, we argue that Pinghua populations did not descend from Han Chinese, but from southern minorities. The ancestral populations of Pinghua people were assimilated by the Han Chinese in terms of language, culture, and self-identification and, consequently, the Pinghua people became an exceptional branch of Han Chinese's coherent genetic structure.