Summary The recently enriched genomic history of Indigenous groups in the Americas is still meager concerning continental Central America. Here, we report ten pre-Hispanic (plus two early colonial) genomes and 84 genome-wide profiles from seven groups presently living in Panama. Our analyses reveal that pre-Hispanic demographic events contributed to the extensive genetic structure currently seen in the area, which is also characterized by a distinctive Isthmo-Colombian Indigenous component. This component drives these populations on a specific variability axis and derives from the local admixture of different ancestries of northern North American origin(s). Two of these ancestries were differentially associated to Pleistocene Indigenous groups that also moved into South America, leaving heterogenous genetic footprints. An additional Pleistocene ancestry was brought by a still unsampled population of the Isthmus (UPopI) that remained restricted to the Isthmian area, expanded locally during the early Holocene, and left genomic traces up to the present day. Highlights • Ancient Isthmian genomes address anthropological questions on pre-contact burials • The comparison with modern Panamanians highlights genomic structure on the Isthmus • A genomic component drives the Isthmian groups on a distinctive variability axis • A previously unknown Pleistocene ancestry identified in the Isthmo-Colombian area Pre-contact and modern genomes from Panama highlight the distinctiveness of the Isthmo-Colombian area; detail number, source, and impact of Indigenous American genomic ancestries at the continental level; and explain complex pre-Hispanic burials. Graphical abstract