The initial colonization of the Americas remains a highly debated topic1, and the exact timing of the first arrivals is unknown. The earliest archaeological record of Mexico—which holds a key geographical position in the Americas—is poorly known and understudied. Historically, the region has remained on the periphery of research focused on the first American populations2. However, recent investigations provide reliable evidence of a human presence in the northwest region of Mexico3,4, the Chiapas Highlands5, Central Mexico6 and the Caribbean coast7–9 during the Late Pleistocene and Early Holocene epochs. Here we present results of recent excavations at Chiquihuite Cave—a high-altitude site in central-northern Mexico—that corroborate previous findings in the Americas10–17of cultural evidence that dates to the Last Glacial Maximum (26,500–19,000 years ago)18, and which push back dates for human dispersal to the region possibly as early as 33,000–31,000 years ago. The site yielded about 1,900 stone artefacts within a 3-m-deep stratified sequence, revealing a previously unknown lithic industry that underwent only minor changes over millennia. More than 50 radiocarbon and luminescence dates provide chronological control, and genetic, palaeoenvironmental and chemical data document the changing environments in which the occupants lived. Our results provide new evidence for the antiquity of humans in the Americas, illustrate the cultural diversity of the earliest dispersal groups (which predate those of the Clovis culture) and open new directions of research. Chiquihuite Cave (Zacatecas, Mexico) provides evidence of human presence in the Americas between about 33,000–31,000 and 14,000–12,000 years ago, and expands the cultural variability known from sites of this date.