<p>Whole Y-chromosome and mitochondrial variations provide insights into deep demographic histories and sex-biased admixture that often remain undetected through autosomal-based analyses. The Nanjing people, an unofficially recognized group in western Guizhou, have historically been described as migrant soldiers from coastal Jiangsu and classified as Bai, exhibiting a complex and poorly understood genetic origin. To investigate their demographic history, we analyzed genetic variation in a large-scale, ethnolinguistically diverse Chinese genomic resource, including 42 newly sequenced genomes from the Nanjing people and 1,454 ancient and modern reference whole-genome sequences. Phylogenetic and demographic analyses revealed that both northern and southern Chinese Y-chromosome lineages contributed to the paternal ancestry of the Nanjing people. We identified one dominant paternal lineage, O2a2b1a1a1c1a1a, that likely originated in northern and eastern China, underwent a bottleneck ~ 1,700 years before present (YBP), and expanded around 600 YBP. In contrast, maternal lineages, particularly F1, M7, and B4, showed strong affinities with southern Chinese and northern Vietnamese populations, indicating a southwestern indigenous maternal origin. The discordant paternal and maternal origin patterns indicate that male migrant soldiers from coastal northern China interbred with indigenous southwestern Chinese females, supporting a strongly sex-biased model of admixture formation. These findings shed new light on the genetic origins and evolution of the Nanjing people, underscoring the power of uniparental markers in disentangling complex and sex-biased demographic processes.</p>