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The ancient city of Chichén Itzá in Yucatán, Mexico, was one of the largest and most influential Maya settlements during the Late and Terminal Classic periods (ad600–1000) and it remains one of the most intensively studied archaeological sites in Mesoamerica1–4. However, many questions about the social and cultural use of its ceremonial spaces, as well as its population’s genetic ties to other Mesoamerican groups, remain unanswered2. Here we present genome-wide data obtained from 64 subadult individuals dating to around ad500–900 that were found in a subterranean mass burial near the Sacred Cenote (sinkhole) in the ceremonial centre of Chichén Itzá. Genetic analyses showed that all analysed individuals were male and several individuals were closely related, including two pairs of monozygotic twins. Twins feature prominently in Mayan and broader Mesoamerican mythology, where they embody qualities of duality among deities and heroes5, but until now they had not been identified in ancient Mayan mortuary contexts. Genetic comparison to present-day people in the region shows genetic continuity with the ancient inhabitants of Chichén Itzá, except at certain genetic loci related to human immunity, including the human leukocyte antigen complex, suggesting signals of adaptation due to infectious diseases introduced to the region during the colonial period.

•The radiocarbon dating results corroborate the use of the pilgrim's shell since at least the 11th century CE.•The data suggest that the pilgrimage was mainly an urban phenomenon for populations from the northern Iberian Peninsula.•The pilgrimage was conducted equally by women and men.•Female pilgrims may have had greater access to animal protein than their male counterparts.•The individuals buried with the scallop shell had δ15N and δ13C indicative of a heterogeneous group.

•Stable isotope analysis of different social strata in Medieval northern Iberia.•Observed differences between historical personages, urban communities, and rural communities.•Expanding urban centres and religious networks between 9th and 13th centuries AD manifested in dietary differences.•Possibilities of novel insights from bioarchaeology even during periods covered by historical records.