Ancient genomes from Mendoza, Argentina, trace the dynamics of colonial urban formation in the Southern Cone of the Americas

European colonization introduced abrupt changes into the Americas. These included the introduction of infectious diseases, forced labor and displacement of Indigenous and African individuals, European settlement, and the imposition of caste regimes. However, the role of these forces in reshaping communities across different regions remains debated. This is especially true in the Virreinato del Río de la Plata (the territories of present-day Argentina, Bolivia, Uruguay, Paraguay, and Brazil), which is less studied than other regions such as the United States, the Caribbean, and Mexico. We investigated how these forces shaped the colonial societies of Mendoza in the Cuyo region (Central-Western Argentina) by sequencing ancient DNA of 28 individuals (17th to 19th centuries) excavated from churches associated with different social strata, recovering 22 mitochondrial genomes, and genome-wide data of 15 individuals. By performing paleogenomic analyses contextualized by burial site, we detected admixture among Indigenous, European, and African populations, with ancestry partially structured by church and burial location. Exceptions to this pattern indicate social mobility and flexible social classifications beyond genetic ancestry. The European-related ancestry aligns with Southern European migration, while African-related ancestry shows affinities to Western-Central Africa. The Indigenous-related ancestry shows highest affinity to precontact populations from Cuyo, supporting local genetic continuity across the colonial period. Additionally, uniparental markers reflect sex-biased dispersal and admixture. These findings reveal often-neglected African contributions to the city's formation and illustrate how caste, labor, and migration shaped colonial urban societies. Our work provides a scalable model for interrogating caste regimes, social mobility, and admixture in understudied regions of the Americas.