AbstractAim  To reconstruct in detail the potential migration routes and barriers for vascular plants from the summits of the Guyana mountains during the Quaternary, in order to test the possibility of migration among them during the glaciations. These changes in connectivity are predicted based on the altitudinal migration of plant communities associated with glacial cooling. To examine the effects of these cycles, the extent of the potential biotic interchange and its influence on patterns of endemism was modelled.Location  The summits of the tepuis or table mountains of the Neotropical Guyana Highlands, which constitute the peculiar and discontinuous Pantepui phytogeographical province (total surface 5000 km2, altitudinal range 1500–3014 m a.s.l.), and is characterized by a unique and diverse flora with a high degree of endemism.Methods  GIS‐based palaeotopographical reconstruction using a high‐precision digital elevation model, combined with phytogeographical analysis by means of a data base built up from the Flora of the Venezuelan Guyana, which includes the geographical and altitudinal ranges for each Pantepui species.Results  During the Last Glacial Maximum, which serves as a representative of a standard Quaternary glaciation, most migration pathways among tepuis were open for species with lower altitudinal levels (LAL) ≤ 1500 m (1678 species or c. 69% of the total Pantepui species), and closed for species with LAL ≥ 2300 m altitude (c. 3%). The species in between these altitudes have intermediate migratory possibilities, depending on the district and the tepui considered. If these local factors are considered, the number of species with no possibility of glacial interchange increases to 202 or c. 8% of the total. The strongest topographical barrier separated eastern locations above 1600–1700 m elevation from all others. The highest possibility of interchange was among the tepuis of the eastern sector, in which internal topographical barriers were only effective for species with LALs at or above 1900 m.Main conclusions  The Quaternary evolution of the vascular flora from the Guyana Highlands took place in a predominantly migration‐prone, glacial‐era landscape, in which more than 70% of the flora (maximum estimate) was able to move from one tepuian district to another, thanks to the downward bioclimatic shift caused by cooling. Interglacials were too short to drive significant evolutionary diversification. A number of present high‐altitude local endemics are species that were unable to migrate, even during glaciations. However, some endemic species do appear to have been able to migrate among regions, suggesting that topographical isolation alone is not enough to explain patterns of endemism. Other factors such as tepui summit area, habitat heterogeneity or pre‐Quaternary evolution are considered. These studies should be complemented with palaeoecological and phylogeographical surveys.